Treating a bacterial skin infection with a whole, leech saliva extract

ABSTRACT

Methods are also provided for isolating and using a whole-saliva leech extract in the treatment of a bacterial skin infection. The methods can include feeding a phagostimulatory agent to a leech; inducing a regurgitation in the leech, the inducing including placing the leech in an environment having a temperature of less than about 0° C.; and, collecting an unrefined, whole saliva in the regurgitation of the cooled leech. The methods can include revitalizing the leech by warming it at a temperature ranging from about 5° C. to about 40° C. Stable, lyophilized, whole-saliva extracts of a leech are also provided, the extract having a stable activity when stored for use at a temperature below about −20° C., the extract maintaining at least 70% of the activity for at least 6 months. The extracts can be used to treat solid tumors, treat liquid tumors, treat diabetes, treat a viral disease, treat a parasitic disease, treat an antibacterial disease, or serve as an anti-oxidant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/991,023, filed Jan. 8, 2016, which is a continuation of U.S.application Ser. No. 14/837,281, filed Aug. 27, 2015, now U.S. Pat. No.9,265,803, which is a continuation of U.S. application Ser. No.14/670,262, filed Mar. 26, 2015, now U.S. Pat. No. 9,144,587, which is acontinuation of U.S. application Ser. No. 14/563,829, filed Dec. 8,2014, now U.S. Pat. No. 9,017,732, which is a continuation of U.S.application Ser. No. 14/282,893, filed May 20, 2014, now U.S. Pat. No.8,962,034, which is a continuation of U.S. application Ser. No.13/624,860, filed Sep. 21, 2012, now U.S. Pat. No. 8,784,896, whichclaims the benefit of U.S. Provisional Application No. 61/701,735, filedon Sep. 17, 2012, each of which is hereby incorporated by reference inits entirety.

BACKGROUND

Field of the Invention

The teachings provided herein are generally directed to methods ofisolating and using a whole-saliva leech extract in the treatment of asubject.

Description of the Related Art

The history of humans using leeches goes back several thousands ofyears, and practically all human civilizations described the use ofleeches to treat different diseases. Unfortunately, due at least to alack of understanding of the chemistries and mechanisms associated withsuch uses, the current state-of-the-art has not been able tosuccessfully commercialize the use of leech saliva extracts in treatingdisease.

There have been attempts at sacrificing leeches to extract activecompounds from the whole body of leeches, from the heads of leeches, orfrom their salivary glands. Much research has been directed toidentifying proteins from leech saliva extracts. None of these efforts,however, have been able to reproduce the effect of using a whole, liveleech, with the exception of, perhaps, the isolation and use of hirudinas an anticoagulant.

There have been attempts at not sacrificing leeches but, rather,extracting a much diluted saliva solution from a live leech.Unfortunately, these efforts have been faced with two major problems:(i) the saliva removal requires a manual squeezing of the leech and, assuch, is not easily scalable; and (ii) the saliva remains dilute, whichcan only be used fresh, and any lyophilization attempts will reduce orcompletely abolish the therapeutic activity of the leech saliva extract.As such, a dose-dependent treatment, or a treatment at elevatedconcentrations, is not available for testing.

One of skill will appreciate (i) a method of isolating an active,refined leech saliva extract (LSE) that can be successfully stored formonths, or even years; (ii) a method of re-using leeches to produce theLSE; (iii) a method of commercializing the isolation and re-use of theleeches to a scalable amount that is practical for commercialization;(iv) a method of treating a solid tumor with the LSE; (v) a method oftreating a liquid tumor with the LSE; (vi) a method of treating diabeteswith the LSE; (vii) a method of treating a virus with the LSE; (viii) amethod of treating a parasitic disease with the LSE; (ix) a method ofusing the LSE as an antioxidant; and (x) a method of using the LSE as anantibacterial.

SUMMARY

The teachings provided herein are generally directed to methods ofisolating and using a whole-saliva leech extract in the treatment of asubject. Pharmaceutical formulations comprising the leech extracts and apharmaceutically acceptable carrier are provided.

The teachings include a method of removing a whole saliva from a leech.In these embodiments, the methods can include feeding a phagostimulatoryagent to a leech; inducing a regurgitation in the leech, the inducingincluding placing the leech in an environment having a temperature ofless than about 0° C.; and, collecting an unrefined, whole saliva in theregurgitation of the cooled leech.

The teachings include a method of creating a lyophilized, whole salivaextract of a leech having an improved stability. In these embodiments,the method can include feeding a phagostimulatory agent to a leech;inducing regurgitation in the leech, the inducing including placing theleech in an environment having a temperature ranging from about −5° C.to about 15° C.; collecting an unrefined, whole saliva in theregurgitation of the cooled leech; removing solid components from theunrefined, whole saliva to create a refined, whole saliva; and,lyophilizing separate volumes of the refined, whole saliva extract, thevolumes not exceeding about 2 ml each.

In some embodiments, the collecting includes squeezing the leech toincrease the amount of unrefined, whole saliva collected. In someembodiments, the methods further comprise revitalizing the leech bywarming the leech in a water bath having a temperature ranging fromabout 5° C. to about 40° C. In some embodiments, the methods furthercomprise creating a refined, whole-saliva extract; the creatingincluding removing solid components from the unrefined, whole saliva. Insome embodiments, the methods further comprise lyophilizing separatevolumes of the refined, whole saliva extract, the volumes not exceedingabout 2 ml each. And, in some embodiments, the leech is Hirudinariamanillensis.

The teachings include a stable, lyophilized, whole-saliva extract of aleech. In these embodiments, the extract comprises a refined,whole-saliva extract of a leech lyophilized in volumes not exceedingabout 2 ml each, the extract refined by removing solid components froman unrefined, whole saliva to create the refined, whole saliva; wherein,the extract has a stable activity when stored for use at a temperaturebelow about −20° C., the extract maintaining at least 70% of theactivity for at least 6 months. And, the leech can be Hirudinariamanillensis.

Methods of treating a subject by administering an effect amount of theleech extracts are provided. In some embodiments, the method includestreating a solid tumor, treating a liquid tumor, treating diabetes,treating a viral disease, treating a parasitic disease, treating abacterial disease, or administering an anti-oxidant therapy. It shouldbe appreciated that each of the treatments also relate to otherconditions that may be desirable to treat in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a method of feeding a phagostimulatory agent to aleech using a membrane, according to some embodiments.

FIGS. 2A-2C illustrate the collection of unrefined, whole salivaextract, according to some embodiments.

FIG. 3 illustrates a UV spectra of the refined, leech saliva extract,according to some embodiments.

FIG. 4 illustrates a standard curve for a colorimetric Bradford proteinassay, according to some embodiments.

FIG. 5 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Laemmli SDS-PAGE 15%gel electrophoresis, according to some embodiments.

FIG. 6 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Laemmli SDS-PAGE 15%gel electrophoresis, wherein the LSE was concentrated using acetoneprecipitation, according to some embodiments.

FIG. 7 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Laemmli SDS-PAGE 15%gel electrophoresis, wherein the LSE was precipitated from solutionusing a trichloroacetic acid (TCA) precipitation, according to someembodiments.

FIG. 8 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Non-Urea SDS-PAGEgel electrophoresis of Okajima, according to some embodiments.

FIG. 9 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Tricine SDS-PAGE gelelectrophoresis method, according to some embodiments.

FIGS. 10A and 10B show the results of RP-HPLC in the analysis of LSE,according to some embodiments.

FIG. 11 shows isolation of LSE proteins using RP-HPLC, according to someembodiments.

FIG. 12 shows the molecular weights of the two isolated proteins usingTricine SDS-PAGE gel electrophoresis, according to some embodiments.

FIG. 13 illustrates IC50 of LSE with respect to antithrombin activity,according to some embodiments.

FIG. 14 shows the relationship between thrombin time and theconcentration of LSE protein, according to some embodiments.

FIG. 15 shows effects of lyophilization conditions and storageconditions on the activity and stability of the LSE, according to someembodiments.

FIG. 16 shows the effect of lyophilization time on antithrombin activityof LSE, according to some embodiments.

FIG. 17 shows the effect of light, and container on antithrombinactivity of LSE samples (lyophilized and non-lyophilized) stored at roomtemperature for up to 7 days, according to some embodiments.

FIG. 18 shows the effect of storage temperature, light, and container onantithrombin activity of LSE samples (lyophilized and non-lyophilized)for up to 180 days at 4° C., according to some embodiments.

FIG. 19 shows the effect of container and lyophilization on antithrombinactivity of LSE samples for up to 180 days at −20° C., according to someembodiments.

FIG. 20 shows that the LSE showed remarkable anti-proliferation activityagainst human small cell lung cancer (SW1271 cell line), according tosome embodiments.

FIGS. 21 and 22 show the cytotoxic effect of mixtures of LSE withirinotecan or carboplatin, according to some embodiments.

FIGS. 23 and 24 show the effect of different doses of LSE and insulin onfasting blood glucose (mmol/L) in normal and diabetic rats at varioustime intervals (h), according to some embodiments.

FIG. 25 shows that the LSE has a prophylactic effect on the onset ofdiabetes, according to some embodiments.

FIGS. 26 and 27 compare the free radical scavenging activity of LSE toL-ascorbic acid (vitamin C), according to some embodiments.

DETAILED DESCRIPTION

The teachings provided herein are generally directed to methods ofisolating and using a whole-saliva leech extract in the treatment of asubject. Pharmaceutical formulations comprising the leech extracts and apharmaceutically acceptable carrier are provided.

It should be appreciated that the term “extract” can be used to refer toa powder form of the compounds of interest, a liquid form of thecompounds of interest, or any one or any combination of the compounds ofinterest in powder or liquid form. One of skill will appreciate that theterm “extract” can be used to refer to the compounds of interest before,during, or after their removal from the leech. In some embodiments, thecompounds of interest can be synthesized chemically using standardmethods known to one of skill, such that they can be synthesized andused alone, or in any combination, by those of skill without use of theextraction methods taught herein. The compositions provided herein canbe referred to as extracts, compositions, compounds, agents, activeagents, bioactive agents, supplements, drugs, and the like. In someembodiments, the terms “LSE,” “extract,” “LSE composition,”“composition,” “compound,” “agent,” “active”, “active agent”, “bioactiveagent,” “supplement,” and “drug” can be used interchangeably and, itshould be appreciated that, a “formulation” can comprise any one or anycombination of these. Likewise, in some embodiments, the composition canalso be in a liquid or dry form, where a dry form can be a powder formin some embodiments, and a liquid form can include an aqueous ornon-aqueous component. Moreover, the terms “activity” or “bioactivity”can refer to the function of the compound in vitro, in an assay forexample, or in vivo when administered to a subject.

It should be appreciated that the leech extracts can be isolated orpurified. In some embodiments, the terms “isolated” and “purified” canbe used interchangeably. In some embodiments, the term “isolated” can beused to refer to the extract being removed from the natural chemicalenvironment of the leech, such that the extract is not in the form inwhich it exists in nature. It should be appreciated that the term“purified” can be used to refer to an extract from a Hirudinariamanillensis leech, in some embodiments, such that the compounds ofinterest are isolated from the remainder of the leech in a form that canbe administered to a subject, such as a soluble form, or a form that cango into aqueous solution. As such, one of skill will appreciate that thecompounds of interest can sometimes be accompanied by other componentsthat are carried along with the extract. For example, such othercomponents can include any one or any combination of proteins found tobe active in the leech. In some embodiments, the term “purified” can beused to refer to an extract consisting of, or consisting essentially of,any one or any combination of the compounds of interest. In someembodiments, the extract includes a phagostimulatory solution or acomponent from the phagostimulatory solution. In some embodiments, anextract “consists essentially of” any one or any combination of thecompounds of interest, where the presence of any other component fromthe leech or extraction procedure has a negligible effect on theactivity of the compounds of interest. The term “negligible effect” canbe used to mean that the activity does not increase or decrease morethan about 10% when compared to any one or any combination of thecompounds of interest, respectively, without the other components. Insome embodiments, the term “negligible effect” can be used to refer to achange of less that 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, and less than 3%. In someembodiments, the term “negligible effect” can be used to refer to achange ranging from about 3% to about 10%, in increments of 1%. Forexample, the activity of the compounds of interest can be enhanced by anamount ranging from about 10% to about 300%, from about 20% to about200%, from about 25% to about 250%, from about 30% to about 300%, fromabout 35% to about 275%, from about 40% to about 225%, from about 15% toabout 100%, or any range therein in increments of 1%.

Methods of removing a whole saliva from a leech are provided. In theseembodiments, the methods can include feeding a phagostimulatory agent toa leech; inducing a regurgitation in the leech, the inducing includingplacing the leech in an environment having a temperature of less thanabout 0° C.; and, collecting an unrefined, whole saliva in theregurgitation of the cooled leech.

One of skill will appreciate that any leech having a therapeutic salivacan be used in the teachings provided herein. In some embodiments, theleech can belong to the family of hirudinidae, to the sub-familyhirudinariiae, or it can belong to a genus selected from the groupconsisting of hirudo; hirudinaria; aliolimantis; limantis;asiaticobdella; goddardobdella; limnobdella; macrobdella; oxyptychus;philobdella. In some embodiments, the leech can be selected from aspecies selected from the group consisting of hirudo medicinalis; hirudotroctina, hirudo nipponia; hirudo orientalis; hirudo verbana;hirudinaria manillensis; hirudinaria javanica; aliolimantis africana;aliolimantis michaelseni; aliolimantis oligodonta; aliolimantisbuntonesis; limantis nilotica; limantis cf. nilotica; limantis paluda;asiaticobdella fenestrata; goddardobdella elegans; limnobdella mexicana;macrobdella decora; macrobdella diploteria; macrobdella diletra;oxyptychus brasiliensis; oxyptychus striatus; philobdella floridana;philobdella gracilis.

In some embodiments, the leech can belong to the family ofhaemadipsidae, or it can belong to a genus selected from the groupconsisting of chtonobdella; haemadipsa; idiobdella; malagdbdella;nesophilaemon. In these embodiments, the leech can be selected from aspecies selected from the group consisting of chtonobdella bilineata;chtonobdella whitmani; haemadipsa interrupta; haemadipsa sylvestris;haemadipsa sumatrana; idiobdella seychellensis; malagdbdella fallax;nesophilaemon skottsbergi.

In some embodiments, the leech can belong to the family ofxerobdellidae, or it can belong to a genus selected from the groupconsisting of diestecostoma; mesobdella; xerobdella. In theseembodiments, the leech can be selected from a species selected from thegroup consisting of diestecostoma magna; diestecostoma mexicana;diestecostoma trujillensis; mesobdella gemmata; xerobdella lecomtei.

In some embodiments, the leech can belong to the family of haemopidae,or it can belong to a genus selected from the group consisting ofhaemopis; whitmania. In these embodiments, the leech can be selectedfrom a species selected from the group consisting of haemopis grandis;haemopis kingi; haemopis sanguisuga; haemopis terrestris; whitmanialaevis.

In some embodiments, the leech can belong to the family ofsemiscolecidae, or it can belong to a genus selected from the groupconsisting of patagoniobdella; semiscolex. In these embodiments, theleech can be selected from a species selected from the group consistingof patagoniobdella fraternal; patagoniobdella variabilis; semiscolexintermedius; semiscolex lamothei; semiscolex similis.

In some embodiments, the leech can belong to the family ofamericobdellidae, or it can belong to a genus selected from the groupconsisting of americobdella. In these embodiments, the leech can beselected from a species selected from the group consisting ofamericobdella valdiviana.

In some embodiments, the leech can belong to the family ofcylicobdellidae, or it can belong to a genus selected from the groupconsisting of cylicobdella. In these embodiments, the leech can beselected from a species selected from the group consisting ofcylicobdella coccinea.

In some embodiments, the leech can belong to the family oferpobdellidae. In these embodiments, the leech can be selected from aspecies selected from the group consisting of erpobdella mentezuma.

The leeches can be classified according to Table 1, in some embodiments.

TABLE 1 Family Sub family Genus Species Hirudinidae HirudinariinaeHirudo Hirudo medicinalis Hirudo nipponia Hirudo orientalis Hirudotroctina Hirudo verbana Aliolimantis Aliolimantis Africana Aliomantismichaelseni Aliomantis oligodonta Aliomantis buntonesis AsiaticobdellaAsiaticobdella fenestrate Goddardobdella Goddardobdella elegansHirudinaria Hirudinaria javanica Hirudinaria manillensis LimantisLimantis nilotica Limantis cf. nilotica Limantis paluda LimnobdellaLimnobdella mexicana Macrobdella Macrobdella decora Macrobdelladiploteria Macrobdella diletra Oxyptychus Oxyptychus brasiliensisOxyptychus striatus Philobdella Philobdella floridana Philobdellagracilis Haemadipsidae Not applicable Chtonobdella Chtonobdellabilineata Chtonobdella whitmani Haemadipsa Haemadipsa interruptaHaemadipsa sylvestris Haemadipsa sumatrana Idiobdella Idiobdellaseychellensis Malagadbdella Malagadbdella fallax NesophilaemonNesophilaemon skottsbergi Xerobdellidae Not applicable DiestecostomaDiestecostoma magna Diestecostoma Mexicana Diestecostoma trujillensisMesobdella Mesobdella gemmata Xerobdella Xerobdella lecomtei HaemopidaeNot applicable Haemopis haemopis grandis Haemopis kingi Haemopissanguisuga Haemopis terrestris Whitmania Whitmania laevis SemiscolecidaeNot applicable Patagoniobdella Patagoniobdella fraternal Patagoniobdellavariabilis Semiscolex Semiscolex intermedius Semiscolex lamotheiSemiscolex similis Americobdellidae Not applicable AmericobdellaAmericobdella valdiviana Cylicobdellidae Not applicable CylicobdellaCylicobdella coccinea Erpobdellidae Not applicable Erpobdella Erpobdellamontezuma

Any phagostimulatory agent known to one of skill can be used. In someembodiments, the phagostimulatory agent can include a protein, apolypeptide, an oligopeptide, or an amino acid. In some embodiments, theamino acid is an L-amino acid selected from the group consisting ofarginine, alanine, leucine, aspartic acid, serine, threonine,isoleucine, histidine, lysine, tryptophan, glycine, phenylalanine,tyrosine, valine, glutamic acid, asparagine, glutamine, cysteine,methionine, and proline. In some embodiments, the phagostimulatory agentis arginine. In some embodiments, the phagostimulatory agent is glycine.In some embodiments, the phagostimulatory agent is proline. In someembodiments, the phagostimulatory agent is a sugar. In some embodiments,the phagostimulatory agent is a sugar selected from the group consistingof fructose, glucose, sucrose, maltose, raffinose, trehalose, robose,and galactose. In some embodiments, the phagostimulatory agent is cornoil. In some embodiments, the phagostimulatory agent comprises any oneor any combination of amino acids and/or sugars taught herein. Anysuitable solvent for carrying the phagostimulatory can be used, polar ornon-polar, as long as the solvent does not substantially affect theactivity or stability of the leech saliva extract.

The temperature of the leech that induces the regurgitation can rangefrom about −5° C. to about 15° C., from about −4° C. to about 14° C.,from about −3° C. to about 13° C., from about −2° C. to about 12° C.,from about −1° C. to about 11° C., from about 0° C. to about 10° C.,from about −2° C. to about 2° C., from about −3° C. to about 3° C., fromabout −4° C. to about 4° C., from about −5° C. to about 5° C., or anytemperature or range of temperatures therein in increments of 1° C. Thetemperature can be established using any method known to one of skill.In some embodiments, the temperature is established to 0° C. or about 0°C. using an ice water bath. In some embodiments, a salt water bath canbe used to lower the temperature below 0° C., and in some embodiments,other liquids can be used to obtain other temperatures. Any method ofcooling know to one of skill can be used to induce the leeches to vomit.The rate of freezing can be 0.1 to 2° C. per minute and, in someembodiments, 1° C. to 1.5° C. per minute. The time at the cooltemperature can vary and can be, for example, from about 5 minutes toabout 45 minutes, from about 15 minutes to about 40 minutes, from about15 minutes to about 20 minutes, from about 10 minutes to about 30minutes, from about 5 minutes to about 25 minutes, from about 3 minutesto about 35 minutes, from about 2 minutes to about 12 minutes, or anytime or range times therein in increments of 1 minute.

Methods of creating a lyophilized, whole saliva extract of a leechhaving an improved stability are provided by the teachings herein. Inthese embodiments, the method can include feeding a phagostimulatoryagent to a leech; inducing regurgitation in the leech, the inducingincluding placing the leech in an environment having a temperatureranging from about −5° C. to about 15° C.; collecting an unrefined,whole saliva in the regurgitation of the cooled leech; removing solidcomponents from the unrefined, whole saliva to create a refined, wholesaliva; and, lyophilizing separate volumes of the refined, whole salivaextract, the volumes not exceeding about 2 ml each.

In some embodiments, the collecting includes squeezing the leech toincrease the amount of unrefined, whole saliva collected. In someembodiments, the methods further comprise revitalizing the leech bywarming the leech in a water bath having a temperature ranging fromabout 5° C. to about 40° C. In some embodiments, the methods furthercomprise creating a refined, whole-saliva extract; the creatingincluding removing solid components from the unrefined, whole saliva. Insome embodiments, the methods further comprise lyophilizing separatevolumes of the refined, whole saliva extract, the volumes not exceedingabout 2 ml each. And, in some embodiments, the leech is Hirudinariamanillensis.

Stable, lyophilized, whole-saliva extracts of a leech are provided bythe teachings herein. In these embodiments, the extract comprises arefined, whole-saliva extract of a leech lyophilized in volumes notexceeding about 2 ml each, the extract refined by removing solidcomponents from an unrefined, whole saliva to create the refined, wholesaliva; wherein, the extract has a stable activity when stored for useat a temperature below about −20° C., the extract maintaining at least70% of the activity for at least 6 months. And, the leech can beHirudinaria manillensis.

Storage temperature has been shown in some embodiments herein to have alarge effect on the stability of the extracts. In some embodiments, forexample, the refined, whole saliva can be stored at a temperatureranging from 0° C. to −80° C., from −20° C. to −270° C., from −20° C. to−196° C., from −20° C. to −80° C., from −80° C. to −196° C., or anytemperature, or any range therein in increments of 1° C.

One of skill will appreciate that the extracts can vary in stability,but that the teachings provided herein show extracts with increasedstabilities when compared to the current state-of-the-art. One of skillwill appreciate that the compositions or formulations should remainstable, or at least substantially stable, until used or activated, andthis can relate to a shelf life, or a time between creation andadministration of the composition, or some combination thereof. In someembodiments, the composition is stable, or substantially stable, whenusable as intended within a reasonable amount of time, a time that isconsidered reasonable by one of skill for the applications taughtherein. In some embodiments, the composition should be usable within areasonable time from the making to the administration of the compositionand, in some embodiments, the composition should have a reasonablecommercial shelf life, a shelf life that is considered reasonable to oneof skill. A reasonable shelf life can be at least 6 months, at least 1year, at least 18 months, at least 2 years, at least 3 years, or anytime in-between in increments of about 1 month, in some embodiments.

In some embodiments, a composition or formulation can be considered as“stable” if it loses less than 10%, less than 7%, less than 6%, lessthan 5%, less than 3%, less than 2%, or less than 1% of its originalactivity. In some embodiments, a composition or formulation can beconsidered as “substantially stable” if it loses greater than about 10%of its original activity, as long as the composition can perform it'sintended use to a reasonable degree of efficacy. In some embodiments,the composition can be considered as substantially stable if it losesactivity at an amount greater than about 12%, about 15%, about 25%,about 35%, about 45%, about 50%, about 60%, or even about 70%. Theactivity loss can be measured by comparing activity at the time ofpackaging to the activity at the time of administration, and this caninclude a reasonable shelf life. In some embodiments, the composition isstable or substantially stable, if it remains useful for a periodranging from 3 months to 3 years, 6 months to 2 years, 1 year, or anytime period therein in increments of about 1 month.

Methods of Treatment

Methods of treating a subject by administering an effect amount of theleech extracts are provided by the teachings herein. The extracts taughtherein can be used for a variety of treatments, preventative,ameliorative, or otherwise, as well as for use as a dietary supplement.The uses can include medicinal purposes, as a health supplement, anutritional composition, a prophylactic, or a treatment of an existingcondition. In some embodiments, any tissue that can make contact withone or more active components of an extract taught herein can betreated. In some embodiments, a tissue can have a desirable secondaryeffect from one or more of the active components of an extract taughtherein making contact elsewhere in the subject, such that one or more ofthe active components can contact a first tissue, whereas a secondtissue realizes a beneficial effect. For example, the first tissue canbe a stomach lining, and the second tissue can realize the desirableeffect of a release of a neurotransmitter or a neuroimpulse. The tissuecan be, for example, connective, muscle, nervous, and/or epithelialtissue. In some embodiments, the tissue is a dermal tissue. In someembodiments, the tissue is a mucosal tissue. And, in some embodiments,the tissue is gastrointestinal tissue. In some embodiments, the methodincludes treating a solid tumor, treating a liquid tumor, treatingdiabetes, treating a viral disease, treating a parasitic disease,administering an anti-oxidant therapy, or administering an antibacterialtherapy.

As such, the subject can have a target tissue that is the focus of thetreatment in which the extracts are applied directly or systemically. Insome embodiments, the term “target site” can be used to refer to aselect location on or in a subject that could benefit from anadministration of a compound taught herein, either parenterally ornon-parenterally, whether injected or administered topically or orally,for example. In some embodiments, a target can include any site ofaction in which the agent's activity can serve a benefit to the subject.The target site can be a healthy or damaged tissue of a subject. Assuch, the teachings include a method of administering one or morecompounds taught herein to a healthy or damaged tissue, dermal, mucosal,gastrointestinal or otherwise.

The terms “treat,” “treating,” and “treatment” can be usedinterchangeably in some embodiments and refer to the administering orapplication of the compositions and formulations taught herein,including such administration as a health or nutritional supplement, andall administrations directed to the prevention, inhibition, ameliorationof the symptoms, or even a cure of a condition taught herein. The terms“disease,” “condition,” “disorder,” and “ailment” can be usedinterchangeably in some embodiments.

The term “subject” and “patient” can be used interchangeably in someembodiments and refer to an animal such as a mammal including, but notlimited to, non-primates such as, for example, a cow, pig, horse, cat,dog, rat and mouse; and primates such as, for example, a monkey or ahuman. As such, the terms “subject” and “patient” can also be applied tonon-human biologic applications including, but not limited to,veterinary, companion animals, commercial livestock, and the like.

Treatment of Cancer

The LSE taught herein can be used in the treatment of cancer. In someembodiments, the methods include treating a solid tumor and, in someembodiments, the methods include treating a liquid tumor. One of skillwill appreciate that the cancers that can be treated using the methodstaught herein can include any hyperproliferative tissue. In someembodiments, for example, any cancer listed in Table 2 can be treatedusing the methods taught herein.

TABLE 2 Cell line Cancer type Cancer Sub-type CCRF-CEM Leukemia AcuteLymphoblastic Leukemia (ALL) HL-60 (TB) Leukemia Acute MyelogenousLeukemia (AML) K-562 Leukemia Chronic Myelogenous leukemia (CML) MOLT-4Leukemia Acute Lymphoblastic Leukemia (ALL) RPMI-8226 MultiplePlasmacytoma, Myeloma myeloma SR Leukemia Acute Lymphoblastic Leukemia(ALL) A549/ATCC Non-small Adinocarcinoma cell lung EKVX Non-smallAdinocarcinoma cell lung HOP-62 Non-small Adinocarcinoma cell lungHOP-92 Non-small Adinocarcinoma cell lung NCI-H226 Non-small SquamousCarcinoma cell lung NCI-H23 Non-small Adinocarcinoma cell lung NCI-H322MNon-small Bronchioloalveolar cell lung Carcinoma NCI-H460 Non-smallAdinocarcinoma cell lung NCI-H522 Non-small Adinocarcinoma cell lungCOLO 205 Colon Adinocarcinoma HCC-2998 Colon Adinocarcinoma HCT-116Colon Carcinoma HCT-15 Colon Adinocarcinoma HT-29 Colon AdinocarcinomaKM12 Colon Colorectal SW-620 Colon Adinocarcinoma SN-268 CNSGlioblastoma SF-295 CNS Glioblastoma SF-539 CNS Gliosarcoma SNB-19 CNSGlioblastoma SNB-75 CNS Glioblastoma LOX IMVI Skin Cancer MelanomaMALME-3M Skin Cancer Melanoma M14 Skin Cancer Melanoma, amelanoticSK-MEL-2 Skin Cancer Melanoma, malignant SK-MEL-28 Skin Cancer Melanoma,malignant SK-MEL-5 Skin Cancer Melanoma, malignant UACC-257 Skin CancerMelanoma UACC-62 Skin Cancer Melanoma IGROVI Ovarian AdinocarcinomaOVCAR-3 Ovarian Adinocarcinoma OVCAR-4 Ovarian Carcinoma OVCAR-5 OvarianCarcinoma OVCAR-8 Ovarian Carcinoma SK-OV-3 Ovarian Adinocarcinoma 786-0Renal Carcinoma A498 Renal Carcinoma ACHN Renal Adinocarcinoma CAKI-1Renal Carcinoma RXF-393 Renal Carcinoma SN12C Renal Carcinoma TK-10Renal Carcinoma UO-31 Renal Carcinoma PC-3 Prostate AdinocarcinomaDU-145 Prostate Carcinoma MCF7 Breast Adinocarcinoma NCI/ADR-RES BreastAdinocarcinoma MDA-MB-231/ATCC Breast Adinocarcinoma HS 578T BreastCarcinosarcoma MDA-MB-435 Breast Carcinoma, ductal MDA-MB-468 BreastAdinocarcinoma BT-549 Breast Carcinoma T-47D Breast Carcinoma, ductal

Treatment of Diabetes

The LSE taught herein can be used in the treatment of diabetes. Examplesof diabetes include Type 1-, Type 2-, and gestational diabetes. As such,one of skill will appreciate that the LSE taught herein can be used intreating and preventing metabolic imbalances, diabetes mellitus, apre-diabetic state, metabolic syndrome, and other related disorders,such as Latent Autoimmune Diabetes in adults (referred to as Type 1.5diabetes). As such, secondary medical conditions related to diabetes canalso be treated using the LSE taught herein, indirectly or directly,including heart disease, stroke, high blood pressure, eye complications(retinopathy, cataracts), kidney disease (nephropathy), nervous systemdisease (neuropathy), peripheral vascular disease, dental disease,gastroparesis, sexual dysfunction, and complications during pregnancy.

The term “diabetic” in a rat can refer to a random blood glucose >225mg/dl or fasting blood glucose level of >110 mg/dL. The term “diabetic”in a human can refer to a random plasma or blood glucose concentrationof ≧200 mg/dL (≧11.1 mmol/L) or a fasting plasma glucose ≧126 mg/dL(≧7.0 mmol/L) or a 2 hour post-load glucose≦200 mg/dL (≧11.1 mmol/L)during an oral glucose tolerance test. The term “non-diabetic” in a ratgenerally means a fasting plasma glucose level of ≦80 mg/dL or a randomplasma glucose level <200 mg/dL. The term “non-diabetic” in a human canrefer to a fasting plasma glucose level of <100 mg/dL (5.6 mmol/dL) or a2 hour post-load glucose<140 mg/dL (<7.8 mmol/dL) during an oral glucosetolerance test. The term “pre-diabetic” in a rat can refer to a fastingplasma glucose level of about 80 to about 110 mg/dL. The term“pre-diabetic” in a human can refer to a fasting plasma glucose level of100-125 mg/dL (5.6-6.9 mmol/L) or a 2 hour post-load glucose 140-199mg/L (7.8-11.1 mmol/L) during an oral glucose tolerance test. The terms“random” and “nonfasting” can be used in reference to any time of day ornight without regard to time since the last meal, and the term “fasting”generally means no caloric intake for at least 12 hours. The term“metabolic imbalance” can refer any condition associated with anelevated plasma glucose. A metabolic imbalance, for example, comprisesdiabetes mellitus, gestational diabetes, genetic defects of .beta.-cellfunction, genetic defects in insulin action, diseases of the exocrinepancreas, endocrinopathies, drug or chemical-induced, infections, othergenetic syndromes associated with diabetes, a pre-diabetic state, andmetabolic syndrome. The term “metabolic syndrome” can refer to a groupof metabolic risk factors in one person including, but not limited to,abdominal obesity, atherogenic dyslipidemia, hypertension, insulinresistance or glucose intolerance, prothrombotic state (high fibrinogenor plasminogen activator inhibitor-1), and proinflammatory state(elevated C-reactive protein). In some embodiments, metabolic syndromebe the presence of three or more of the following components: elevatedwaist circumference (males: ≧40 inches, females ≧35 inches), fastingtriglycerides ≧150 mg/dL, reduced HDL (males: <40 mg/dL, females<50mg/dL), blood pressure ≧130/85 mm Hg, and fasting glucose ≧100 mg/dL.

The above definitions for diabetes follow standards of the AmericanDiabetes Association (ADA), the American Heart Association (AHA) and theNational Heart, Lung, and Blood Institute. Other definitions can be usedand may vary by region or country, and may depend upon the group orinstitution (e.g. ADA, World Health Organization (WHO), NationalInstitute of Diabetes and Digestive and Kidney Diseases (NIDDK/NIH),Center for Disease Control (CDC), etc.) providing other guidelines.Physicians may also use clinical experience, a patient's past medicalhistory, and the like when deciding on a diagnosis and treatment. Assuch, one of skill will appreciate that the particular ranges andmeasures are merely relative rather than critical to making a diagnosisor planning a treatment. In some embodiments, for example, any of theabove measures can vary by about 1%, about 2%, about 3%, about 5%, about7%, about 10%, about 15%, about 20%, about 25%, about 30%, 40%, 50%, orany range or amount therein in increments of 0.1%.

Treatment of a Viral Disease

The LSE taught herein can be used in the treatment of several differenttypes of viral diseases. In some embodiments, the virus can be a speciesof Adenoviridae, Herpesviridae, Papillomaviridae, Polyomaviridae,Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae,Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Retroviridae,Orthomyxoviridae, Arenaviridae, Bunyaviridaem, Filoviridae,Paramyxoviridae, Rhabdoviridae, or Reoviridae.

In some embodiments, the species of virus treated can be selected fromthe group consisting of Adenovirus, Herpes simplex, type 1, Herpessimplex, type 2, Varicella-zoster virus, Epstein-barr virus, Humancytomegalovirus, Human herpesvirus, type 8, Human papillomavirus, BKvirus, JC virus, Smallpox, Hepatitis B virus, Human bocavirus,Parvovirus B19, Human astrovirus, Norwalk virus, coxsackievirus,hepatitis A virus, poliovirus, rhinovirus, Severe acute respiratorysyndrome virus, Hepatitis C virus, yellow fever virus, dengue virus,West Nile virus, Rubella virus, Hepatitis E virus, and Humanimmunodeficiency virus (HIV).

In some embodiments, the viral condition can be a regionally identifiedcondition selected from the viral conditions in Table 3:

TABLE 3 United Australia Hong Kong Malaysia Kingdom United StatesAcquired Acquired Immunodeficiency immunodeficiency Syndrome (AIDS)syndrome Arbovirus Arbovirus Arbovirus infections: infections:infections: California serogroup Barmah Forest, West Nile virus, Easternequine Dengue fever, virus encephalitis virus, Japanese Powassan virus,St. encephalitis, Louis encephalitis Kunjin virus, virus, West Nilevirus, Murray Valley Western equine encephalitis virus, encephalitisvirus Ross River virus Chickenpox Chickenpox (i.e., varicella)-morbidityand deaths only Chikungunya fever Dengue Dengue fever Dengue fever feverEnterovirus 71 infection Hantavirus Hantavirus infection HepatitisHepatitis Hepatitis Hepatitis A Hepatitis A Hepatitis A Hepatitis BHepatitis B Hepatitis B Hepatitis C Hepatitis C Hepatitis C Hepatitis DHepatitis D Hepatitis E Hepatitis E Human Human HIV infectionimmunodeficiency immunodeficiency virus (HIV) virus (HIV) infectioninfection Influenza Influenza A Influenza-associated (H2), pediatricmortality and Influenza A novel influenza A (H5), infection Influenza A(H7) or Influenza A (H9) Japanese encephalitis Lyssavirus MeaslesMeasles Measles Measles Measles Mumps Mumps Mumps Mumps PoliomyelitisAcute Poliomyelitis Poliomyelitis Poliomyelitis, poliomyelitis paralyticand non- paralytic Rabies Rabies Rabies Rabies Rubella Rubella andRubella Rubella congenital rubella syndrome Severe Severe Severe AcuteAcute Acute Respiratory Respiratory Respiratory Syndrome SyndromeSyndrome Smallpox Smallpox Smallpox Smallpox Yellow fever Yellow feverYellow fever Yellow fever Yellow fever Viral Viral Viral Viralhemorrhagic hemorrhagic haemorrhagic hemorrhagic fever, including feverfever, including fever Arenavirus (new Lassa fever, world), Crimean-Marburg virus, Congo hemorrhagic and Ebola virus fever, Denguehemorraghic fever, Ebola virus, Lassa virus, Marburg virus

In some embodiments, the compositions taught herein can be administeredwith a second agent, such as abacavir, aciclovir, acyclovir, adefovir,amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla,aoceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine,docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,entry inhibitors, famciclovir, fomivirsen, fosamprenavir, foscarnet,fosfonet, fusion inhibitor, ganciclovir, ibacitabine, imunovir,idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor,interferon type III, interferon type II, interferon type I, interferon,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a,penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor.Raltegravir, reverse transcriptase inhibitor, ribavirin, rimantadine,ritonavir, pyramidine, saquinavir, stavudine, synergistic enhancer(antiretroviral), tea tree oil, telaprevir, tenofovir, tenofovirdisoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada,valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine,zalcitabine, zanamivir, and zidovudine.

Treating a Parasitic Disease

The LSE taught herein can be used in the treatment of several differenttypes of parasitic diseases. In some embodiments, the parasitic diseasetreated can be classed as a condition caused by protozoa (causingprotozoan infection), helminths (helminthiasis), and ectoparasites.

In some embodiments, the parasitic disease can be selected from thegroup consisting of Acanthamoeba keratitism, Amoebiasis, Ascariasis,Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease,Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis,Dracunculiasis (caused by the Guinea worm), Echinococcosis,Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis,Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayamafever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis,Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleepingsickness, Strongyloidiasis, Taeniasis (cause of Cysticercosis),Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

In some embodiments, the compositions taught herein can be administeredwith a second agent, such as thiabendazole, pyrantel pamoate,mebendazole, praziquantel, niclosamide, bithionol, oxamniquine,metrifonate, Ivermectin, albendazole, benznidazole, nifurtimox, andnitroimidazole.

Treatment of a Bacterial Disease

The LSE taught herein can be used in the treatment of several differenttypes of bacterial diseases. In some embodiments, the bacterial diseasecan include, for example, tuberculosis from Mycobacterium tuberculosis;pneumonia from Streptococcus and Pseudomonas; a foodborne illness fromShigella, Campylobacter, or Salmonella; and, either tetanus, typhoidfever, diphtheria, syphilis, or leprosy. In some embodiments, thebacterial disease can be a bacterial vaginosis; bacterial meningitis;bacterial pneumonia; urinary tract infection, including E. coli.Infections; bacterial gastroenteritis, also including E. coli; and,bacterial skin infections, including impetigo from S. aureus and S.pyogenes, Erysipelas from Streptococcus, and cellulitis which caninclude connective tissue. In some embodiments, the bacterial diseasecan be selected from the group consisting of the diseases in Table 4.

TABLE 4 United Australia Hong Kong Malaysia Kingdom United StatesAnaplasmosis Anthrax Anthrax Anthrax Botulism Botulism Botulism BotulismBrucellosis Brucellosis Brucellosis Campylobacteriosis ChancroidChlamydia Chlamydia trachomatis Cholera Cholera Cholera Cholera CholeraDiphtheria Diphtheria Diphtheria Diphtheria Diphtheria DonovanosisEhrlichiosis Shiga toxin-and Escherichia coli Escherichia verocytotoxin-O157:H7 coli O157:H7 producing infection or Shiga-toxin Escherichia coliproducing (STEC/VTEC) Escherichia coli Encephalitis EncephalitisGonococcal Gonococcal Gonorrhea infection infection/Gonorrhea HaemolyticHaemolytic Hemolytic uraemic syndrome uraemic uremic (HUS) syndromesyndrome, (HUS) post-diarrheal Haemophilus Haemophilus Haemophilusinfluenzae serotype influenzae type influenzae, b (Hib) b infectioninvasive (invasive) disease Legionellosis Legionnaire's Legionnaire'sLegionellosis Disease Disease Leprosy Leprosy Leprosy Leprosy Hansen'sdisease (Leprosy) Leptospirosis Leptospirosis Listeriosis ListeriosisListeriosis Lyme disease Meningococcal Meningococcal MeningococcalMeningococcal disease infection septicaemia/ disease (invasive) AcuteMeningitis MSRA: Community- associated methicillin- resistantStaphylococcus aureus infection Paratyphoid Paratyphoid feverParatyphoid fever fever Pertussis Pertussis Pertussis Pertussis(Whooping cough) (Whooping (Whooping cough) (Whooping cough) cough)Plague Plague Plague Plague Plague (bubonic, (bubonic, septicemic,septicemic, pneumonic and pneumonic pharyngeal) and pharyngeal)Psittacosis Psittacosis Psittacosis Q fever Q fever Q Fever, acute andchronic Relapsing fever Relapsing fever Rickettsiosis Rickettsiosis,spotted fever Scarlet fever Scarlet fever Salmonellosis SalmonellosisShigellosis Bacillary Shigellosis dysentery Group A Group AStreptococcal Streptococcal disease disease Pneumococcal Streptococcusdisease pneumoniae, invasive disease Streptococcus suis infectionSyphilis Syphilis Syphilis Tetanus Tetanus Tetanus Tetanus Tetanus Toxicshock syndrome (Streptococcal and other than Streptococcal) TuberculosisTuberculosis Tuberculosis Tuberculosis Tuberculosis, Mycobacteriumtuberculosis Tularemia Tularemia Typhoid fever Typhoid fever Typhoidfever Typhoid fever Typhoid fever Typhus and Typhus Typhus otherrickettsial diseases Vancomycin Intermediate Staph Aureus (VISA),Vancomycin Resistant Staph Aureus (VRSA)

Administering an Anti-Oxidant Therapy

The LSE taught herein can be used in antioxidant therapy. One of skillwill appreciate that reactive oxygen species (ROS) are widely believedto cause or aggravate several human pathologies such as arthritis,neurodegenerative diseases, cancer, heart disease, stroke and many otherailments. Antioxidants can be used to counteract the harmful effects ofROS and therefore prevent or treat oxidative stress-related diseases. Insome embodiments, the LES taught herein can be used as a free radicalscavenger, or to prevent oxidation in the body. In some embodiments, theLES taught herein can be used to treat inflammatory disorders, endocrinedisorders, cardiovascular disease, aging, as well as to serve as aneuroprotective agent. In some embodiments, the LES taught herein can beused to treat atherosclerosis. And, in some embodiments, the LES can beadministered in combination with a cholesterol medication such as anabsorption blocker, a synthesis inhibitors and a niacin-based drug. Insome embodiments, a non-drug alternative can be used, such asbeta-glucan from whole oats or barley; psyllium from wheat bran; or,phytosterols and/or phytostanols.

In some embodiments, the absorption blocker can be cholestyramine orZETIA. In some embodiments, the synthesis inhibitor can be a statinincluding, but not limited to, MEVACOR, PRAVACHOL, ZOCOR, LIPITOR,LESCOL, CRESTOR, or LIVALO. In some embodiments, the synthesis inhibitorcan be LOVASTATIN, PRAVASTATIN, or SIMVASTATIN. In some embodiments, theniacin-based medication can be NIASPAN or NIACOR. In some embodiments,the cholesterol medication can be a combination product such as MEVACORwith NIASPAN, or ZETIA with ZOCOR.

Methods of Administration

Any administration vehicle known to one of skill to be suitable foradministration of the compounds, compositions, and formulations taughtherein can be used. A “vehicle” can refer to, for example, a diluent,excipient or carrier with which a compound is administered to a subject.

The terms “administration” or “administering” can be used to refer to amethod of incorporating a composition into or onto the cells or tissuesof a subject, either in vivo or ex vivo to test the activity of asystem, as well as to diagnose, prevent, treat, or ameliorate a symptomof a disease or condition. In one example, a compound can beadministered to a subject in vivo using any means of administrationtaught herein. In another example, a compound can be administered exvivo by combining the compound with cell tissue from the subject forpurposes that include, but are not limited to, assays for determiningutility and efficacy of a composition. And, of course, the compositionscan be used in vitro to test their stability, activity, toxicity,efficacy, and the like. When the compound is incorporated in the subjectin combination with one or active agents, the terms “administration” or“administering” can include sequential or concurrent incorporation ofthe compound with the other agents such as, for example, any agentdescribed above. A composition can be formulated, in some embodiments,to be compatible merely with its intended route of administration.

Any dosage form known to one of skill can be used for administrationsthat include, for example, parenteral and non-parenteraladministrations. In some embodiments, the composition is in a dosageform for administration topically. And, in some embodiments, thecomposition is in a dosage form for administration orally. In someembodiments, the dosage form can be a capsule or an injectable fluid.The composition can also be used as a dietary supplement. The term“dosage unit” can refer to discrete, predetermined quantities of acompound that can be administered as unitary dosages to a subject. Apredetermined quantity of active compound can be selected to produce adesired therapeutic effect and can be administered with apharmaceutically acceptable carrier. The predetermined quantity in eachunit dosage can depend on factors that include, but are not limited to,(a) the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of creating and administering such dosage units.

A “pharmaceutically acceptable carrier” is a diluent, adjuvant,excipient, or vehicle with which the composition is administered. Acarrier is pharmaceutically acceptable after approval by a state orfederal regulatory agency or listing in the U.S. PharmacopeialConvention or other generally recognized sources for use in subjects.The pharmaceutical carriers include any and all physiologicallycompatible solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. Examples of pharmaceutical carriers include, but are not limitedto, sterile liquids, such as water, oils and lipids such as, forexample, phospholipids and glycolipids. These sterile liquids include,but are not limited to, those derived from petroleum, animal, vegetableor synthetic origin such as, for example, peanut oil, soybean oil,mineral oil, sesame oil, and the like.

Suitable pharmaceutical excipients include, but are not limited to,starch, sugars, inert polymers, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol, and the like. In some embodiments, thecomposition can also contain minor amounts of wetting agents,emulsifying agents, pH buffering agents, or a combination thereof. Oralformulations, for example, can include standard carriers such as, forexample, pharmaceutical grades mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike. See Martin, E. W. Remington's Pharmaceutical Sciences.

As described herein, the compositions can take the form of lotions,creams, suspensions, emulsions, tablets, pills, capsules, powders,sustained-release formulations and the like. In some embodiments, thecompositions or formulations can be administered to a subject in anynon-parenteral manner known to one of skill whereas, in contrast, aparenteral administration involves piercing the skin or a mucousmembrane. Depending on the target tissue, the administration can betopical, oral, ocular, otologic, nasal, urogenital, rectal, dermal,vaginal or otherwise to a mucous membrane. Oral administration, forexample, can include digestive tract, buccal, and sublingualadministration, and a solid or liquid carrier can be used. One of skillwill appreciate that the therapeutic program selected, the agentsadministered, the condition of the subject, and the effects desired, canaffect the administration schedule and program used.

The compositions or formulations can be contained in forms that includetablets, troches, capsules, elixirs, beverages, suspensions, syrups,wafers, chewing gums, gels, hydrogels, and the like. Tablets, pills,capsules, troches liquids and the like may also contain binders,excipients, disintegrating agent, lubricants, glidants, chelatingagents, buffers, tonicity modifiers, surfactants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Some examples of excipientsinclude starch or maltodextrin. Some examples of disintegrating agentsinclude alginic acid, corn starch and the like. Some examples oflubricants include magnesium stearate or potassium stearate. An exampleof a chelating agent is EDTA. Some examples of buffers are acetates,citrates or phosphates. Some examples of tonicity modifiers includesodium chloride and dextrose. Some examples of surfactants formicellation or increasing cell permeation include coconut soap, anionic,cationic or ethoxylate detergents. An example of a glidant is colloidalsilicon dioxide. Some examples of sweetening agents include sucrose,saccharin and the like. Some examples of flavoring agents includepeppermint, chamomile, orange flavoring and the like.

In the digestive tract, for example, a solid can include a pill,capsule, tablet, or time-release technology in some embodiments; and, aliquid can include a solution, soft gel, suspension, emulsion, syrup,elixir, tincture, or a hydrogel. Digestive tract administration caninclude oral or rectal administration using any method known to one ofskill. For buccal, sublingual, and sublabial administration, a solid caninclude an orally disintegrating tablet, a film, a lollipop, a lozenge,or chewing gum; and, a liquid can include a mouthwash, a toothpaste, anointment, or an oral spray.

One of skill understands that the amount of the agents administered canvary according to factors such as, for example, the type of disease,age, sex, and weight of the subject, as well as the method ofadministration. Dosage regimens may also be adjusted to optimize atherapeutic response. In some embodiments, a single bolus may beadministered; several divided doses may be administered over time; thedose may be proportionally reduced or increased; or, any combinationthereof, as indicated by the exigencies of the therapeutic situation andfactors known to one of skill in the art. It is to be noted that dosagevalues may vary with the severity of the condition to be alleviated, aswell as whether the administration is prophylactic, such that thecondition has not actually onset or produced symptoms. Dosage regimensmay be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and any dosage ranges set forthherein are exemplary only and do not limit the dosage ranges that may beselected.

An “effective amount” of a compound can be used to describe atherapeutically effective amount or a prophylactically effective amount.An effective amount can also be an amount that ameliorates the symptomsof a disease. A “therapeutically effective amount” can refer to anamount that is effective at the dosages and periods of time necessary toachieve a desired therapeutic result and may also refer to an amount ofactive compound, prodrug or pharmaceutical agent that elicits anybiological or medicinal response in a tissue, system, or subject that issought by a researcher, veterinarian, medical doctor or other clinicianthat may be part of a treatment plan leading to a desired effect. Insome embodiments, the therapeutically effective amount should beadministered in an amount sufficient to result in amelioration of one ormore symptoms of a disorder, prevention of the advancement of adisorder, or regression of a disorder. In some embodiments, for example,a therapeutically effective amount can refer to the amount of an agentthat provides a measurable response of at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100% of a desired action of thecomposition.

In cases of the prevention or inhibition of the onset of a disease ordisorder, or where an administration is considered prophylactic, aprophylactically effective amount of a composition or formulation taughtherein can be used. A “prophylactically effective amount” can refer toan amount that is effective at the dosages and periods of time necessaryto achieve a desired prophylactic result, such as prevent the onset of asunburn, an inflammation, allergy, nausea, diarrhea, infection, and thelike. Typically, a prophylactic dose is used in a subject prior to theonset of a disease, or at an early stage of the onset of a disease, toprevent or inhibit onset of the disease or symptoms of the disease. Aprophylactically effective amount may be less than, greater than, orequal to a therapeutically effective amount.

In some embodiments, a therapeutically or prophylactically effectiveamount of a composition may range in concentration from about 0.01 nM toabout 0.10 M; from about 0.01 nM to about 0.5 M; from about 0.1 nM toabout 150 nM; from about 0.1 nM to about 500 μM; from about 0.1 nM toabout 1000 nM, 0.001 μM to about 0.10 M; from about 0.001 μM to about0.5 M; from about 0.01 μM to about 150 μM; from about 0.01 μM to about500 μM; from about 0.01 μM to about 1000 nM, or any range therein. Insome embodiments, the compositions may be administered in an amountranging from about 0.005 mg/kg to about 100 mg/kg; from about 0.005mg/kg to about 400 mg/kg; from about 0.01 mg/kg to about 300 mg/kg; fromabout 0.01 mg/kg to about 250 mg/kg; from about 0.1 mg/kg to about 200mg/kg; from about 0.2 mg/kg to about 150 mg/kg; from about 0.4 mg/kg toabout 120 mg/kg; from about 0.15 mg/kg to about 100 mg/kg, from about0.15 mg/kg to about 50 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, orany range therein, wherein a human subject is often assumed to averageabout 70 kg.

In some embodiments, the compositions or formulations can beadministered in conjunction with at least one other therapeutic agentfor the condition being treated. The amounts of the agents can bereduced, even substantially, such that the amount of the agent or agentsdesired is reduced to the extent that a significant response is observedfrom the subject. A “significant response” can include, but is notlimited to, a reduction or elimination of a symptom, a visible increasein a desirable therapeutic effect, a faster response to the treatment, amore selective response to the treatment, or a combination thereof. Insome embodiments, the other therapeutic agent can be administered, forexample, in an amount ranging from about 0.1 μg/kg to about 1 mg/kg,from about 0.5 μg/kg to about 500 μg/kg, from about 1 μg/kg to about 250μg/kg, from about 1 μg/kg to about 100 μg/kg from about 1 μg/kg to about50 μg/kg, or any range therein. Combination therapies can beadministered, for example, for 30 minutes, 1 hour, 2 hours, 4 hours, 8hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 6weeks, 3 months, 6 months, 1 year, 2 years, any combination thereof, orany amount of time considered desirable by one of skill. The agents canbe administered concomitantly, sequentially, or cyclically to a subject.Cycling therapy involves the administering a first agent for apredetermined period of time, administering a second agent or therapyfor a second predetermined period of time, and repeating this cyclingfor any desired purpose such as, for example, to enhance the efficacy ofthe treatment. The agents can also be administered concurrently. Theterm “concurrently” is not limited to the administration of agents atexactly the same time, but rather means that the agents can beadministered in a sequence and time interval such that the agents canwork together to provide additional benefit. Each agent can beadministered separately or together in any appropriate form using anyappropriate means of administering the agent or agents. One of skill canreadily select the frequency, duration, and perhaps cycling of eachconcurrent administration.

Each of the agents described herein can be administered to a subject incombination therapy. In some embodiments, the agents can be administeredat points in time that vary by about 15 minutes, 30 minutes, 1 hour, 2hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, 48 hours or 1week in time. In some embodiments, at least one of the agents is animmunomodulatory agent. In other embodiments, the agents can includeantiproliferatives, antineoplastics, antimitotics, anti-inflammatories,antiplatelets, anticoagulants, antifibrins, antithrombins, antibiotics,antiallergics, antioxidants, and any prodrugs, codrugs, metabolites,analogs, homologues, congeners, derivatives, salts and combinationsthereof.

Without intending to be limited to any theory or mechanism of action,the following examples are provided to further illustrate the teachingspresented herein. It should be appreciated that there are severalvariations contemplated within the skill in the art, and that theexamples are not intended to be construed as providing limitations tothe claims.

Example 1 A Method of Removing a Whole Saliva from a Leech

This example shows that leeches can be fed a phagostimulatory agent,induced to regurgitate the agent to collect the whole saliva as anunrefined, whole saliva in the regurgitation, and then be revitalizedfor reprocessing to collect more saliva. The regurgitation can beinduced, for example, by significantly lowering the leeches bodytemperature to a state of paralysis or near-paralysis to induce avomiting. The leeches can then be warmed to re-animate, or revitalize,the leeches for storage and/or the reprocessing to collect more saliva.

The leeches were collected by a local supplier from the natural lake,Cheneh, located in Terengganu, Malaysia. The leeches were maintained atroom temperature under 12 h:12 h light and dark cycle in well-aeratedplastic containers filled with un-chlorinated tap water which wasregularly changed every 2-3 days.

FIG. 1 illustrates a method of feeding a phagostimulatory agent to aleech using a membrane, according to some embodiments. As shown in FIG.1, the leeches 105 were fed a solution of the phagostimulatory agent 110comprising 0.001M arginine in normal saline. The leeches 105 were fedusing the feeding device having the parafilm membrane 120 stretchedacross the glass funnel 100 filled with the phagostimulatory solution110 warmed at a temperature of 37° C. The starved leeches 105 attach tothe membrane 120, feed by sucking the phagostimulatory solution 110through the membrane 120 until satiated, and drop spontaneously.

FIGS. 2A-2C illustrate the collection of unrefined, whole salivaextract, according to some embodiments. The engorged leeches 105 thatwere fed the phagostimulatory solution 110 were transferred topolypropylene containers 205 as shown in FIG. 2A, immersed in an icebath 210 for about 15 to about 20 minutes as shown in FIG. 2B, andinduced to vomit an unrefined, whole saliva 215 as shown in FIG. 2C.

The low temperature induced a regurgitation of the phagostimulatorysolution 110, as well as a sort of paralysis or near-paralysis of theleech 105. The paralyzed leeches 105 were squeezed to remove additionalunrefined whole saliva 215 without harming the leeches 105. A valuableprocess consideration is that the leeches 105 were found to readilyregain their activity by immersing them in a warm water bath at 37° C.for about 15 to about 30 min, after which they are revitalized and canbe stored for re-use.

The unrefined whole saliva was a colorless fluid that was pooled andcentrifuged at 4° C. and 9000 rpm for 15 min to remove solids and refinethe whole saliva. To further refine the whole saliva, the supernatantwas filtered using a 0.45 μm filter paper. The refined leech salivaextract was aliquoted in amber flat-bottom glass tubes in amounts thatdid not exceed 2 ml for a 24-hour lyophilization cycle. Beforelyophilization, the refined extracts were frozen at −80° C. for 30 min.After lyophilizations, the refined extracts were kept at −80° C. in theclosed, amber flat-bottom glass tubes.

Example 2 Chemical Characterization of the Leech Saliva Extract

This example provides a chemical characterization of the refined, leechsaliva extract (LSE).

Standard procedures known to those of skill were used to produce UVspectra of the LSE. The spectra were obtained by scanning and measuringthe λ_(max), showing an optimum protein spectrum with 2 λ_(max) valuesat 199 nm and 207 nm.

FIG. 3 illustrates a UV spectra of the refined, leech saliva extract,according to some embodiments. The spectra of leeches' saliva extractwere determined using UV spectrophotometer in the following steps: a) UVlamp was warmed up for about 15 min, b) the instrument was adjusted tospectrum mode, c) wavelengths were adjusted to a λ_(min)=190 nm, and aλ_(max)=800 nm, d) a blank (the phagostimulatory solution) was used tocalibrate to zero.

Standard procedures known to those of skill were used to produce aquantitative colorimetic proten assay, in which a reagent kit havingbovine serum albumin (BSA) as a standard protein was used. Bradford, M.M. Anal. Biochem. 72: 248-254(1976). A phagostimulatory solution having0.001M arginine in 0.15M NaCl was used as a blank, and a series ofknown-concentrations of BSA (10 μg/ml to 250 μg/ml) were prepared in thephagostimulatory solution. Three dilutions of the LSE were prepared inthe phagostimulatory solution, and 100-μl volumes of the BSA, LSE andblank were aliquoted in EPPENDROF tubes with an equal volume of Bradfordreagent and mixed well. The absorbance at 595 nm (A₅₉₅) were measuredusing a microplate reader. The A₅₉₅ values of the blank were subtractedfrom those of BSA and LSE, and a standard curve of the knownconcentrations of BSA against their A₅₉₅ values was prepared todetermine total protein concentration of the leech saliva extract fromthe plot.

FIG. 4 illustrates a standard curve for a colorimetric Bradford proteinassay, according to some embodiments. The standard curve wasY=0.001X−0.011, where: X=BSA concentration (μg/ml) and Y=absorbance at595 nm, R²=0.993. It was found that the total protein concentration ofthe colorless LSE collected from leeches starved for 16 weeks was119.691±8.690 μg/ml, whereas leeches starved for 22-weeks yielded LSEwith a total protein concentration of 62.682±2.459 μg/ml. Table 2describes the total protein concentration results of LSE collected fromleeches starved for 16 and 22 weeks as the mean of triplicates,expressed as the mean±standard deviation SD (n=3).

TABLE 2 Absorbance A₅₉₅ Replicate Replicate Replicate 1 2 3 A₅₉₅ BSAconc. (μg/ml)   12.5 0.007 0.006 0.000 0.004 ± 0.004  25 0.017 0.0240.021 0.021 ± 0.003  50 0.074 0.067 0.069 0.070 ± 0.003 100 0.120 0.1230.127 0.124 ± 0.003 125 0.170 0.172 0.173 0.171 ± 0.002 150 0.194 0.2050.204 0.201 ± 0.006 Blank Arg/NaCl (μl) 100 0.287 0.285 0.277 0.283 ±0.005 LSE volume (μl) Starvation 80 0.080 0.087 0.099 0.088 ± 0.010period 16 90 0.095 0.112 0.098 0.102 ± 0.009 weeks 100 0.098 0.099 0.0990.099 ± 0.000 Starvation 80 0.037 0.045 0.040 0.041 ± 0.004 period 22 900.044 0.042 0.043 0.043 ± 0.001 weeks 100 0.053 0.056 0.048 0.052 ±0.004 Total protein concentration in LSE (μg/ml) Starvation period124.333 125.074 109.667 119.691 ± 8.690  16 weeks (November) Starvationperiod 64.750 59.963 63.333 62.682 ± 2.459  22 weeks (December)

Standard gel electrophoresis procedures known to those of skill wereused to produce molecular weight distributions of the LSE. Theseparation of molecules within a gel is determined by the relative sizeof the pores formed within the gel. The pore size of a gel is determinedby two factors, the total amount of acrylamide present (designated as %T) and the amount of cross-linker (% C). As the total amount ofacrylamide increases, the pore size decreases.

Laemmli SDS-PAGE Gel Electrophoresis of LSE

The Laemmli SDS-PAGE gel electrophoresis method is commonly used andknown to one of skill in the art. The method is widely-used to separateproteins based on electrophoretic mobility.

STOCK SOLUTIONS AND BUFFERS: Stock solutions and buffers were preparedfor a Laemmli SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel) gelelectrophoresis as follows:

In preparing stock solutions, an acrylamide/bisacrylamide (30% T, 2.67%C) (AB 30) was prepared, calculating % C and % T according to (Hjerten,1962):

${{{\%\mspace{14mu} T} = \frac{{g\mspace{14mu}{acrylamide}} + {g\mspace{14mu}{bisacrylamide}}}{100\mspace{14mu}{ml}\mspace{14mu}{solution}}};{and}},{{{\%\mspace{14mu} C} = {\left( \frac{g\mspace{14mu}{acrylamide}}{{g\mspace{14mu}{acrylamide}} + {g\mspace{14mu}{bisacrylamide}}} \right) \times 100}};}$

such that 29.2 g acrylamide 29.2 g and 0.8 g bisacrylamide weredissolved in distilled water and the volume was brought to 100 ml in avolumetric flask. The solution was filtered by using WHATMAN filterpaper grade 1 under vacuum. The solution was kept in a dark container at4° C. A 10% (w/v) SDS was prepared by dissolving 10 g of SDS in 90 mlwater with gentle stirring and the volume was brought to 100 ml withdistilled water in a volumetric flask. The solution was kept in roomtemperature.

In preparing the 10% APS (ammonium persulfate; prepared and used freshdaily) stock solution as a polymerization initiator, 100 mg of APS wasdissolved in 1 ml of distilled water and used immediately.

In preparing a 1.5M tris-HCl, pH 8.8 buffer, 18.15 g of Tris base(tris(hydroxymethyl)aminomethane) was dissolved in 80 ml distilledwater, and the pH was adjusted to 8.8 with 6N HCl. The total volume wasbrought to 100 ml with distilled water and stored at 4° C.

In preparing a 0.5 M tris-HCl buffer, pH 6.8, 6 g of the Tris base wasdissolved in 60 ml distilled water. The pH was adjusted to 6.8 with 6 NHCl. The total volume was brought to 100 ml with distilled water andstore at 4° C.

In preparing an SDS reducing buffer (sample buffer), 1.25 ml of 0.5Mtris-HCl was mixed with 2.5 ml glycerol, 2 ml of the 10% SDS, and 0.2 mlof 0.5% (w/v) bromophenol blue. The total volume was brought to 10 mlwith distilled water in a volumetric flask. The buffer was stored atroom temperature. 50 μl β-mercaptoethanol were added to 950 μl samplebuffer at the time of use.

In preparing a 10× electrode (running) buffer, pH 8.3, 30.3 g of theTris base, 144.0 g glycine, and 10.0 g SDS were dissolved in distilledwater under gentle stirring and the last volume was brought to 1 literwith distilled water. The buffer was kept at room temperature. Whenrunning the gel, 100 ml of this buffer were taken and the volume wasbrought to 1 liter.

MAKING THE GEL: The gel electrophoresis procedure was run using a miniprotein tetra cell BIO RAD instrument. The gel (6×8 cm×1 mm) wasprepared using glass plates, a gel caster, a resolving gel, and astacking gel as follows:

In preparing a resolving gel 15%, 5 ml of the acrylamide/bisacrylamidestock solution, 2.4 ml distilled water, 2.5 ml of the pH 8.8 tris bufferand 0.1 ml of the SDS stock solution were mixed and degassed for about15 min, and 50 μl of the APS stock solution and 5 μl of TEMED (N, N,N′,N′-tetramethylethylenediamine) were added.

In preparing the stacking gel, 1.7 ml of the acrylamide/bisacrylamidestock solution, 5.7 ml distilled water, 2.5 ml of the pH 6.8 trisbuffer, and 0.1 ml SDS were mixed and degassed for about 15 min. 50 μlof the APS stock solution and 10 μl of TEMED were added.

The resolving gel was poured into the gel slabs using a plastic syringeand 1.5 cm over the separating gel was left empty for the stacking gel.100 μl isopropanol were laid on the surface of the gel for smoothnessand to avoid dehydration, and the gel was allowed to polymerize forabout 45 minutes. The isopropanol was removed after polymerization ofthe resolving gel, and stacking gel was added after washing the surfaceof the resolving gel with a separating gel buffer. A comb was added toform cells, and the stacking gel was allowed to polymerize for about 30minutes, and the comb was removed from the gel. The cells were washedwith the electrophoresis buffer, and the gel slab was placed in theelectrophoresis tank, and the tank was filled with the electrophoresisbuffer.

PREPARING THE LSE: the LSE was lyophilized as described herein, and theLSE powder was dissolved in a sample buffer and heated at 95° C. for 5min in a water bath. SDS was added to the sample buffer to help in thedenaturation of proteins, masking the surface of proteins with negativecharges to balance the charge/size ratio for all proteins, such that theseparation will be based only on the size of the protein. Heating theprotein samples before loading helps in completely denaturing allproteins, increases solubility and reduction of disulfide reductionwithout degradation of proteins (Voerman, 1998).

RUNNING THE GEL: the sample was applied to the cells using amicropipette, and a peptide marker was applied to one cell. Theelectrophoresis lid was placed carefully, the electrodes were attachedto a power source, and the electrophoresis was run for 35 minutes at200V.

Coomassie Brilliant Blue dye was used to visualize proteins anddetermine molecular weights from the polyacrylamide gels. A 1 L stockdye solution was prepared by dissolving 1 g Coomassie Brilliant BlueR-250 in 450 ml methanol and 100 ml glacial acetic acid. Distilled waterwas added to increase total volume to 1 L. The stock dye solution wasfiltered using WHATMAN filter paper grade 1 and kept at roomtemperature. A 1 L destaining solution was prepared by mixing 100 mlmethanol with 100 ml glacial acetic acid and adding distilled water toincrease the total volume to 1 L.

After electrophoresis, the gel was transferred to a plastic containercontaining stock dye solution and left there for 30 minutes. Thestaining solution was discarded and the gel was incubated in thedestaining solution for 30 minutes with agitation. This step wasrepeated three to four times with fresh destaining solution, and the gelwas incubated in destaining solution overnight. The gel was imaged anddocumented using a BIO RAD gel imager.

Non-Urea SDS-PAGE Gel Electrophoresis of LSE for Peptides

This portion of the gel electrophoresis analysis was performed accordingto the Okajima method, which is considered to give better results forpeptides. (Okajima, et al., 1993). The method generally uses the samestock solutions and buffers as the Laemmli SDS-PAGE method, an exceptionbeing the separating gel buffer.

SEPARATING/STACKING GEL BUFFER: a 3M tris-HCl, pH 8.45 buffer was madeby dissolving 36.3 g of the Tris base in distilled water, pH wasadjusted to 8.45 with 6N HCl, and the total volume was brought to 100 mlwith distilled water.

MAKING THE GEL: In preparing the resolving (separating) gel 19.2%, 10 mlof the AB 30 stock solution was mixed with 3.75 ml of the separatingbuffer, 0.15 ml SDS, and 1 ml water. The mixture was degassed for 15 minusing the sonicator, and 50 μl of the APS stock solution and 10 μl ofthe TEMED stock solution were added. The mixture was poured into the gelslabs using a plastic syringe and allowed to polymerize for 45 minutes.In preparing the stacking gel 4%, 1.3 ml of the AB 30 stock solution wasmixed with 2.5 ml stacking gel buffer, 0.1 ml SDS, and 6 ml water. Themixture was degassed for 15 minutes before adding APS 50 μl and TEMED 10μl. The sample buffer used for the Laemmli method above was used forthis method.

PREPARING THE LSE AND RUNNING THE GEL: the LSE was lyophilized asdescribed herein, and the LSE powder was dissolved in a sample bufferand heated at 95° C. for 5 min in a water bath. SDS was added to thesample buffer to help in the denaturation of proteins, masking thesurface of proteins with negative charges to balance the charge/sizeratio for all proteins, such that the separation will be based only onthe size of the protein. 20 μl of the sample was applied for the gel.The gel was run for 100 minutes at 100V. Commassie blue staining wasused to stain the gel.

Tricine SDS-PAGE Gel Electrophoresis of LSE for Peptides in the Range of1-100 kDa

This portion of the gel electrophoresis analysis was performed accordingto a tricine SDS-PAGE method commonly used to separate proteins in thesmaller molecular weight range of 1-100 kDa, and preferably used forresolving proteins smaller than 30 kDa. The use of tricine instead ofglycine as a reduction agent provides a better separation of peptideshaving such low molecular weights.

STOCK SOLUTIONS AND BUFFERS: The AB 30 stock solution, 10% (w/v) SDS,10% (w/v) APS, and the sample buffer (SDS reducing buffer) is the sameas that used in the Laemmli SDS-PAGE method described herein; and, theseparating (stacking) gel buffer of Okajima method is used. Otherwise,this method generally uses the same stock solutions and buffers as theLaemmli SDS-PAGE method.

A 10× cathode buffer was prepared by dissolving 12.1 g Tris base,tricine, and 1 g SDS in distilled water. The total volume was brought to100 ml, and the solution was kept at room temperature. The buffer wasdiluted 10 times before use. In addition, a 10× anode buffer wasprepared by dissolving 12.1 g Tris base in water and adjusting pH to 8.9with HCl 6 N. The total volume was brought to 100 ml, and the solutionwas kept at room temperature. The buffer was diluted 10 times beforeuse.

A fixation solution of 5% glutaraldehyde was prepared by add 10 ml of a50% glutaraldehyde to distilled water and bringing the total volume to100 ml. The solution was filtered using WHATMAN filter paper grade 1under a fume hood and used fresh.

MAKING THE GEL: The gels were made according to methods known in theart. (Schägger & von Jagow, 1987). In preparing the resolving(separating) gel 16%, 5 ml of AB 30 was mixed with 5 ml of theseparating buffer, 1.5 ml glycerol, and 1.5 ml distilled water. Themixture was degassed for 15 minutes, and 50 μl of the APS stock solutionand 5 μl of the TEMED stock solution were added. The gel was poured tothe gel slab without delay. The surface of gel was covered by 100 μlisopropanol and allowed to polymerize for 45 minutes. In preparing thestacking gel 4%, 1 ml of the AB 30 was mixed with 3 ml gel buffer, and11 ml distilled water. The mixture was degassed for 15 minutes, and 100μl of the APS stock solution and 10 μl of the TEMED stock solution wereadded. Without delay, the gel was poured to the gel slab. The comb waspositioned, and the gel was allowed to polymerize for 30 minutes.

PREPARING THE LSE AND RUNNING THE GEL: the LSE was lyophilized asdescribed herein, and the LSE powder was dissolved in a sample bufferand heated at 95° C. for 5 min in a water bath. SDS was added to thesample buffer to help in the denaturation of proteins, masking thesurface of proteins with negative charges to balance the charge/sizeratio for all proteins, such that the separation will be based only onthe size of the protein. 20 μl of the sample was applied for the gel.The gel was run for 5 hours, running at 40V for the first 3 hours andincreasing voltage by 10V every 30 minutes. After electrophoresis, thegel was washed with distilled water for 5 minutes and repeated threetimes. The washed gel was transferred to a container of the fixersolution for 1 hour and washed with distilled water to remove theglutaraldehyde. The fixed gel was placed in Commassie blue stainingsolution for 30 min with gentle agitation. The destaining solution wasapplied for 30 min with agitation, and this step was repeated severaltimes until the band became clear.

The Gel Electrophoresis Results

FIG. 5 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Laemmli SDS-PAGE 15%gel electrophoresis, according to some embodiments. Lane 1 is thepeptide marker, and lanes 1-4 represent the week number at which thesaliva was extracted in duplicate; wherein, lanes 1-1′ are week 2, lanes2-2′ are week 3, lanes 3-3′ are week 4, and lanes 4-4′ are week 0. Ascan be seen, the method works well, as the results showed goodresolution with highly isolated bands.

FIG. 6 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Laemmli SDS-PAGE 15%gel electrophoresis, wherein the LSE was concentrated using acetoneprecipitation, according to some embodiments. This method showed a highresolution and clear bands, with a protein molecular weight distributionranging from 10812 Da to 88210 Da. Lanes 1 and 2 are LSE, and lane 4 isthe peptide marker.

FIG. 7 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Laemmli SDS-PAGE 15%gel electrophoresis, wherein the LSE was precipitated from solutionusing a trichloroacetic acid (TCA) precipitation, according to someembodiments. The results show clear bands with a high resolution,although acetone precipitation gave better resolution for the proteinsbands. Lanes 2 and 3 are LSE, and lane 1 is the peptide marker.

FIG. 8 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Non-Urea SDS-PAGEgel electrophoresis of Okajima, according to some embodiments. Smallermolecular weight peptides and proteins were shown having good resolutionwith clear bands. The molecular weight range is wider compared to theclassic Laemmli SDS-PAGE method, as proteins as small as 6.5 kDa weredetected. Lanes 2 and 3 are LSE, and lane 1 is the peptide marker.

FIG. 9 shows the LSE protein molecular weight distribution results of aMalaysian leech, Hirudinaria manillensis, using the Tricine SDS-PAGE gelelectrophoresis method, according to some embodiments. The resultsshowed more than 20 proteins and peptides ranging in molecular weightfrom 4276 Da to 44386 Da. Lanes 2 and 3 are LSE, and lane 1 is thepeptide marker.

The data compared well to known literature values of Hirudinariaspecies, such as bufridin (7 kDa), manillase (58 kDa), hirullin P18 (6.8kDa) and gelin (8.2 kDa). The data suggested other proteins may beshared with other species, such as Calin (65 kDa), Destabilase lysozym(12 kDa), lefaxin (30 kDa), Hirudin (7 kDa) and hyaloronidase (28.5kDa).

Reverse-Phase HPLC of LSE

This example shows how to use analytical chromatography (Buffer (A),0.1% TFA in water and Buffer (B), 0.1% TFA in acetonitrile) of the crudesaliva extract to identify more than 30 peaks with high resolution inthe LSE. In particular, reverse-phase HPLC (RP-HPLC) can be used.

MATERIALS AND METHODS: An Agilent C₁₈ RP column, buffer (A) 0.1% TFA inwater, buffer (B) 0.1% TFA in acetonitrile, a 1 ml/min flow rate, and a5% gradient: 5% (B) over 5 min, 5-90% (B) over 40 min wavelength 214 nm,A lyophilized saliva, B fresh saliva. The lyophilized saliva extractafter reconstitution in distilled was applied to the C₁₈ RP column at aflow rate of 1 ml/min, and a gradient of 5% of (B) over 5 min, followedby 5%-90% (B) over 40 min, and then 90% of (B) over 5 min, and finally90%-5% of (B) over 5 min. The UV detector was set at 214 nm, and avolume 100 μl was injected in the loop. A blank (0.15M saline+0.001Marginine) was run before each analysis.

FIGS. 10A and 10B show the results of RP-HPLC in the analysis of LSE,according to some embodiments. As shown, the results were the same, orat least substantially similar, for lyophilized (FIG. 10A) and fresh(FIG. 10B) saliva extracts.

FIG. 11 shows isolation of LSE proteins using RP-HPLC, according to someembodiments. As can be seen, 30 peaks were isolated from the LSE.Examples of two isolated proteins from the LSE are indicated by arrows.

FIG. 12 shows the molecular weights of the two isolated proteins usingTricine SDS-PAGE gel electrophoresis, according to some embodiments.Lane 1 is the peptide marker, lane 2 is protein 2, and lane 3 isprotein 1. The molecular weights of the two isolated proteins, protein 1and protein 2, were 6289.799 Da and 14244.58 Da, respectively.

Anticoagulant Activity of LSE

This example shows that (i) the lyophilized LSE retains anticoagulantactivity, and (ii) active components of the LSE can be identified usingknown methods. The LSE was frozen at −40° C., lyophilized, dissolved in60 μl distilled water, and used to assess the anticoagulant activity ofisolated portions of the LSE. Isolated proteins were identified andassessed for anticoagulant activity, and the results revealed two activeproteins that extend thrombin time. They were given the names. “protein1” and “protein 2” and prolonged thrombin time by 26.23% and 31.65%,respectively. The isolated proteins were also assessed for inhibition ofamidolytic activity of thrombin, and the results show that theyinhibited the amidolytic activity of thrombin by 30.61% and 41.22% forprotein 1 and protein 2, respectively, confirming the results obtainedregarding thrombin time.

The determination of the amidolytic activity of LSE was based on itsinhibitory effect on thrombin-induced release of p-nitroanilide from thesynthetic substrate of thrombin S-2238 using known methods. (Mao et al.,1987; Schmied, Hoeffken, Hornberger, & Bernard, 1995).

Materials and Methods

-   1. Preparation of the reaction buffer: All reagents used for this    experiment were prepared in phosphate buffered saline-bovine serum    albumin buffer (PBS-BSA, pH 7.4) which contains 0.12M NaCl, 0.01M    sodium phosphate, 0.01% NaN₃ and 0.1% bovine serum albumin.-   2. Thrombin reagent and thrombin substrate S-2238: were prepared in    PBS-BSA to a final concentration of 0.6NIHU thrombin/ml and 100 μM,    respectively. Thrombin substrate solution was preserved at −20° C.    to be used within one month according to storage conditions provided    by the manufacturer.-   3. Amidolytic assay procedures: Volumes of 50 μl of thrombin reagent    were mixed with equal volumes of different dilutions of LSE in the    96-well plate. The plate was shaken gently and incubated for 10 min    at 25° C. in the microplate reader. Thereafter, 100 μl of the    substrate was pipetted and the mixture was agitated. The absorbance    at 405 nm (A₄₀₅) was monitored for eight hours at 5-minute    intervals. Same procedures were done using the phagostimulatory    solution (PhS) as a negative control. Reaction buffer PBS-BSA was    considered as a control.-   4. Calculations: All measurements were repeated in triplicates and    the means were considered. The percentage inhibition (% inhibition)    was calculated from the equation:

${\%\mspace{14mu}{inhibition}} = {\left( \frac{{{Absorbance}\mspace{14mu}{of}\mspace{14mu}{control}} - {{Absorbance}\mspace{14mu}{of}\mspace{14mu}{LSE}}}{{Absorbance}\mspace{14mu}{of}\mspace{14mu}{control}} \right) \times 100}$

FIG. 13 illustrates IC50 of LSE with respect to antithrombin activity,according to some embodiments. The LSE effectively inhibitedthrombin-mediated release of the p-nitroanilide from the syntheticsubstrate (S-2238). The protein concentration that inhibits 50% ofthrombin activity (IC₅₀) was determined by plotting the % inhibitionagainst total protein concentration in the LSE, and it was found to be49.391±2.219 μg/ml. The dose responsive curve of the amidolytic activityof leech saliva extract. Y=2.28X+38.26, where: Y=% inhibition andX=protein concentration (μg/ml), R²=0.878.

Antithrombin activity was determined using a thrombin time (TT) assay invitro. The following standard protocols were used as provided withTHROMBOCLOTIN reagent and a SYSMIX CA 50 COAGULOMETER:

-   -   1. Citrated plasma preparation: prepared from fresh human blood        taken by venipuncture immediately prior to the experiment. Fresh        human blood (4.5 ml) was mixed with sodium citrate in a citrate        tube containing 0.5 ml of 0.11 mol/l sodium citrate (9 parts of        blood: 1 part of sodium citrate). The mixture was centrifuged at        room temperature (25° C.) for 10 min at 3000 rpm. The        supernatant citrated plasma was kept at room temperature (+25 C)        to be used within four hours of preparation.    -   2. Thrombin reagent preparation: Each vial of THROMBOCLOTIN was        reconstituted with 10.0 ml distilled water. The resulted        solution contains 2.5NIHU thrombin/ml and was stable for one        week when stored at 2° C.-8° C.    -   3. Control plasma preparation: a control plasma test was used        before each experiment to evaluate the precision and accuracy of        the reagents used and the coagulometer. One vial of CONTROL N (a        control plasma used to test the instrument) was dissolved in 1.0        ml distilled water, shaken gently and let to stand for 15        minutes at room temperature. The reconstituted control plasma        was kept at −20° C. for a maximum period of four weeks.    -   4. Thrombin time assay: An aliquot 100 μl of the prepared        citrated plasma was pipetted into the pre-warmed coagulation        tube provided with the coagulometer and subsequently incubated        at 37° C. in the coagulation analyzer well for 3 minutes. 100 μl        of the reconstituted thrombin reagent (2.5NIHU thrombin/ml) was        added and the time until coagulation started was measured by the        coagulometer. Different dilutions of the fresh LSE were mixed        with the freshly prepared citrated plasma to yield a final        volume of 100 μl and TT values of the mixtures were measured.        The phagostimulatory solution was used as a negative control.    -   5. Calculations: All measurements were repeated in triplicates        and the means were considered. The percentage increase of        thrombin time (% TT) was calculated from the equation:

${\%\mspace{14mu}{TT}} = {\left( \frac{{{TT}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{sample}} - {{TT}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{citrated}\mspace{14mu}{plasma}}}{{TT}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{citrated}\mspace{14mu}{plasma}} \right) \times 100}$

Fresh LSE collected from leeches starved for 16 weeks prolonged thrombintime (TT) of the citrated plasma in a dose dependent manner. Leechsaliva protein concentration which can increase TT two-fold (IC₁₀₀) wasestimated by plotting % TT values against saliva protein concentrationsthat were mixed with the citrated plasma.

FIG. 14 shows the relationship between thrombin time and theconcentration of LSE protein, according to some embodiments. Theconcentration of LSE protein which increased TT two-fold (IC₁₀₀) wasestimated from the curve of saliva protein concentration (μg/ml plasma)versus percentage increase of TT (% TT). Consequently, it was found thatIC₁₀₀ was 22.558 μg/ml plasma. The results show that the antithromboticactivity of LSE was a linear function with the protein concentration inplasma, Y=4.953X−11.73, where: Y=% TT and X=protein concentration(μg/ml), R²=0.984.

Example 3 A Method of Creating a Stable, Lyophilized, Whole-SalivaExtract of a Leech

This example shows the substantial effect of lyophilization conditionsand storage conditions on the activity and stability of the LSE.Antithrombin activity was used as a measure of the activity andstability of the LSE under the different conditions. Lyophilizationconditions such as vessel type, pre-freezing temperature, lyophilizationtime, and storage conditions were all varied to determine their effectson LSE activity.

The LSE was aliquoted in separate glass and polypropylene tubes eachcontaining 1 ml. The samples were then frozen at −20° C. or −40° C., andthe frozen samples were lyophilized for 12, 24, 48 or 72 hours. Theantithrombin activity (% TT) of each lyophilized sample was determinedand compared with that of the fresh LSE. In addition, glass orpolypropylene tubes, each containing 1 ml of lyophilized ornon-lyophilized LSE were stored at room temperature, 4° C., and −20° C.Some tubes at room temperature were protected from light by wrappingthem with aluminum foil. The antithrombin activity (% TT) of each samplewas monitored for a period of six months and compared with that of thefresh LSE.

FIG. 15 shows effects of lyophilization conditions and storageconditions on the activity and stability of the LSE, according to someembodiments. The results are the mean of triplicates±the standard errorof the mean SEM (n=3), analyzed using one-way ANOVA and Tukey's HSD posthoc test; p<0.05 was considered statistically significant. Freezing at−40° C. before lyophilization significantly (p<0.05) decreased theantithrombotic activity of LSE by 31-34% when compared to the activityof fresh LSE. Freezing at −20° C. before lyophilization provided anantithrombin activity (% TT=60-65%) similar to that of fresh LSE (%TT=62%), regardless the vessel type. The container had no significanteffect on LSE activity during lyophilization.

FIG. 16 shows the effect of lyophilization time on antithrombin activityof LSE, according to some embodiments. All samples were lyophilized for24 h in glass tube. ^(α)p<0.001 when compared with fresh LSE. Theresults are the mean of triplicates±the standard error of the mean SEM(n=3), analyzed using one-way ANOVA and Tukey's HSD post hoc test;p<0.05 was considered statistically significant. Lyophilization for morethan 24 hours led to a dramatic decrease 67-80% (p<0.001) inantithrombin activity. Lyophilization for 12-24 hours, on the otherhand, retained about 95% of its original activity.

Storage at Room Temperature

After one day of storage, all samples (lyophilized or non-lyophilized)stored at room temperature over time lost activity compared to theinitial activity of fresh LSE.

Fresh samples stored in glass tubes exposed to light lost more than 90%activity after one day. Non-lyophilized LSE kept in glass tubesprotected from light lost 62.2% activity after one day of storage, andmore than 90% after 3 days of storage. Non-lyophilized samples kept inpolypropylene tubes showed more than 90% loss of activity after astorage period of one day, regardless of protection from light.Lyophilized LSE kept in glass tubes protected from light for one, three,and seven days lost about 26.5%, 75% and 95% activity, respectively.Lyophilized LSE exposed to light for one day lost about 48% activity,and about 90% after 3-7 days. Lyophilized LSE kept in polypropylenetubes in the dark for one day lost 57% activity, and more than 90%activity after 3 days. Lyophilized LSE kept in polypropylene tubes andexposed to light lost 80%-99% activity over 7 days.

Light significantly affected LSE activity at room temperature.Lyophilized LSE kept in glass tubes protected from light lost 26.5%activity in one day compared to a 48% (p<0.05) activity loss for samplesexposed to light. Non-lyophilized samples kept in polypropylene tubesprotected from light lost 62.2% activity after one day, and lost about92% (p<0.001) of their activity when exposed to light.

The type of the container affected activity of LSE when stored at roomtemperature. Lyophilized samples stored in glass tubes lost 26.5%-47.8%activity, whereas those stored in polypropylene tubes lost 57.1%-84.5%(p<0.05) activity. Non-lyophilized samples kept in glass tubes protectedfrom light lost 62% activity, whereas such samples stored inpolypropylene tubes lost 92% (p<0.001) activity in one day protectedfrom light.

Lyophilization provided stability to the LSE at room temperature.Non-lyophilized samples show a substantial loss of activity whencompared to lyophilized samples when stored under the same conditions.Non-lyophilized LSE stored in glass tubes protected from light lost62.2% activity after one day, whereas the lyophilized LSE lost 26.5%activity after one day (p<0.001). After 3-7 days of storage, significantdifferences between samples were not observed because samples lost agreat part of their biological activity (75-95%).

FIG. 17 shows the effect of light, and container on antithrombinactivity of LSE samples (lyophilized and non-lyophilized) stored at roomtemperature for up to 7 days, according to some embodiments. The resultsare the mean±standard error of the mean SEM (n=3) and analyzed byGeneral Linear Model (GLM), repeated measure ANOVA, using SPSS 18.0software, and p<0.05 was considered statistically significant. α issignificant when compared with fresh LSE (reference control); β issignificant when compared with lyophilized LSE stored in glass tubes andprotected from light; γ is significant when compared with lyophilizedLSE stored in polypropylene tubes and protected from light; c issignificant when compared with non-lyophilized LSE stored in glass tubesand protected from light; and δ is significant when compared withlyophilized LSE in glass tubes in light.

Storage at 4° C.

At a reduced temperature of 4° C., no significant loss of activityoccurred in seven days, regardless of sample type or storage conditions.However, all samples showed a significant decrease (p<0.001) in activityafter 15 days when compared to the initial activity of fresh saliva(control reference).

Non-lyophilized samples kept in glass tubes retained 100%-97% activityduring the seven days. A sharp decline (45%) in activity occurred after15 days, and longer storage times showed more than a 90% loss ofactivity. Lyophilized samples kept in glass tubes retained about 100%activity after seven days, lost about 27% of activity after 15 days, andlost about 80-90% activity after 30 days.

Non-lyophilized samples kept in polypropylene containers retained100-95% activity during the seven days, lost about 47% activity after 15days, and more than 90% activity after 30 days. Lyophilized salivasamples kept in polypropylene containers lost about 0-9% activity after3 days, about 13% after 7 days, about 32% after 15 days, and about85-95% after 30 days.

FIG. 18 shows the effect of storage temperature, light, and container onantithrombin activity of LSE samples (lyophilized and non-lyophilized)for up to 180 days at 4° C., according to some embodiments. After 30days-180 days, all samples lost 81-98% of their activity. The resultsare the mean±standard error of the mean SEM (n=3) and analyzed byGeneral Linear Model (GLM), repeated measure ANOVA, using SPSS 18.0software, and p<0.05 was considered statistically significant. α issignificant when compared with fresh LSE (reference control); β issignificant when compared with lyophilized LSE stored in glass tubes; γis significant when compared with lyophilized LSE stored inpolypropylene tubes. The type of container only had a minor effect,whereas lyophilization had a significant effect on activity after 15days of storage. Non-lyophilized samples showed much more activity lossthan lyophilized samples. Non-lyophilized samples kept in glass tubeslost 45% activity, while lyophilized counterparts lost 27% after 15 daysof storage (p<0.05-0.001). Non-lyophilized samples kept in polypropylenetubes lost 47% compared to a loss of 32% in lyophilized samples (p<0.05)for the same period of 15 days.

Storage at −20° C.

At the storage temperature of −20° C., the type of container and stateof the extract were not statistically significant. Non-lyophilized LSEstored in glass tubes lost from 0-6% activity (statisticallyinsignificant) in 15 days at −20° C. After 30 days, about 10% activitywas lost. After 90-180 days, a significant loss of about 12-15% (p<0.05)activity was observed. Non-lyophilized LSE kept in polypropylene tubeslost 0-5% (statistically insignificant) activity in 15 days, and about13-16% (p<0.05) after 30-180 days. Lyophilized LSE stored in glass tubeslost only 0-5% activity in 180 days (statistically insignificant).Lyophilized samples stored in polypropylene tubes lost about 3-6%activity in 15 days and about 13%-20% (statistically significant) after30-180 days.

FIG. 19 shows the effect of container and lyophilization on antithrombinactivity of LSE samples for up to 180 days at −20° C., according to someembodiments. The results are the mean±standard error of the mean SEM(n=3) and analyzed by General Linear Model (GLM), repeated measureANOVA, using SPSS 18.0 software, and p<0.05 was considered statisticallysignificant.

Example 4 A Method of Treating a Solid Tumor

This example shows the cytotoxic activity of the LSE prepared accordingto Example 1 in the treatment of a solid tumor.

REAGENTS: all reagents prepared as desired under strict sterileconditions per ESCO Class II Biological Safety Cabinet; Leibovitz's L-15medium (from Sigma-Aldrich); phosphate buffered saline (PBS), 1× sterilesolution (from Amresco); L-glutamine (L-Glu, liquid, 200 mM),penicillin/streptomycin (pen/strep, 100×), fetal bovine serum FBSmycoplex and ACCUTASE (a combination of protease and collagen in PBSwith 0.5 mM EDTA) (from The Cell Culture Company PAA); trypan blue dye(from Merck); and CELLTITER-GLO luminescent cell viability assay (fromPromega); bovine serum albumin and arginine hydrochloride (fromSigma-Aldrich); sodium chloride (from Merck); Bradford reagent kit (fromAmresco); carboplatin (cis-Diamine[1,1-cyclobutanedicarboxylato]platinum II) (from Calbiochem); Irinotecanhydrochloride (USP reference standard from Rockville, Md.).

EQUIPMENT: a Jouan CR22 refrigerated centrifuge (Jouan, France); aMemmert incubator type BE-400 (Memmert, Germany); an inverted microscope(from Olympus model CK30); a TECAN microplate luminometer (TECAN, USA);an Infinite M200, NanoQuant TECAN multi detection microplate reader(from TECAN (USA)); and a Christ freeze-drier model Alpha 1-4LD(Germany).

Methods

A human small cell lung cancer (SW1271 cell line) was obtained from theAmerican Type Cell Collection ATCC. According to ATCC standardprotocols, the anchorage dependent cell line was cultivated at aninitial inoculums cell concentration of 10⁴ cells/cm² in 15 ml completegrowth media (CGM) which consists of Leibovitz's L-15 mediumsupplemented with 10% FBS(v/v), 0.3 g/L of L-Glu, and 1% (v/v) pen/strepin a CORNING 75 cm² canted neck cell culture flask. The cultivated cellswere incubated at 37° C. in CO₂-free humidified atmosphere. The CGM wasstored at +4° C. and warmed (37° C.) for 15 min in a water bath prior tousage (ATCC, 2007). Flasks containing the cultivated cells were checkedat 24 h intervals for cell viability, adherence, morphology andconfluence state using the inverted microscope. Cultures were examinedfor any macroscopic evidence of microbial contamination by the invertedmicroscope. Media was changed as needed when media color turns toyellow, as Leibovitz's L-15 medium contains red phenol which becomesyellow at low pH levels and bright red at pH 7.4 which is suitable forcell culture (ATCC, 2007).

When the monolayer of anchorage-dependent cell line SW1271 is near 90%confluent, they were subcultured according to protocols provided by theATCC. After aspirating the CGM from the flasks, the adherent cells weredissociated from the cell culture flask walls by pipetting 3 mlACCUTASE. After an incubation period of 15 minutes with ACCUTASE at 37°C., cells were examined under the inverted microscope to be sure thatmost (95%) cells were detached and dispersed into a single-cellsuspension (ATCC, 2007).

Counting the viable cells was done using trypan blue dye exclusion whichdepends on counting the unstained cells that have not uptake the dyeappearing rounded with halos following the below protocol (NSF, 2006):

-   -   1. Trypan blue solution was prepared in sterile BPS to a final        concentration of 0.4% (w/v).    -   2. Cell suspension was diluted by a sterile BPS to a total        volume of 4 ml so that cell do not overlay on each other making        counting difficult and inaccurate.    -   3. Both hemocytometer and coverslip, were cleaned, dried and        assembled.    -   4. Cell suspension and trypan blue was mixed thoroughly at a        ratio of 1:1 (creating a dilution factor of 2). Thence, 10 μl of        the mixture was pipetted into the counting chamber of the        hemocytometer. Touching the tips with the edge of the coverslip        is sufficient to fill the chamber because of the capillary        action.    -   5. Cell number in the square on each corner was counted and the        average was considered.    -   6. The total cell number was estimated using the following        equation:        Total cell number=average count per square×dilution        factor×10⁴×the total volume of the diluted cell suspension

Cell suspension was homogenized by gentle pipetting and then dispensedat a final density of 10⁴ cells/cm² into new cell culture flaskscontaining 15 ml of CGM. The flasks were regularly monitored to checkfor cell viability and microbial contamination (ATCC, 2007).

When cells reached roughly 90% confluence, they were harvested asdescribed above using ACCUTASE as a dissociating agent. The ACCUTASE wasremoved by gentle centrifugation (10 min, at +4° C. and 125×g) with therefrigerated centrifuge, the supernatant was discarded, and cells werere-suspended in 4 ml of CGM. The cells were counted using the trypanblue dye exclusion, and 10⁴ cells were seeded into a CORNING COSTAR96-well flat bottom cell culture microplate containing 200 μl of CGMusing 8-channel EPPENDORF micropipettor. The microplates were incubatedat 37° C. in a free-CO₂ humidified environment for 24 hours (ATCC,2007).

After the 24-hour incubation, the medium was discarded and replaced bynew 180 μl of CGM. A series of double dilutions of the concentratedlyophilized leech saliva extract (10×LSE) was prepared. The 10×LSE wasfiltered through 0.2 μm SARTORIUS sterile filter paper and 20 μlaliquots were added to the first three rows of the microplate with thehigher concentration in the first row and so forth making the totalvolume 200 μl (180 μl of CGM+20 μl of 10×LSE). To the next three rows,20 μl volumes of another double dilution series of a ten-timeconcentrated of the phagostimulatory solution were added. Anothernegative control plate was prepared containing untreated cells (10⁴cells/well) cultivated in 200 μl of CGM.

Other plates were prepared following the same protocols by replacing10×LSE by carboplatin and irinotecan as positive controls with a serialtwo-fold dilution of both starting from 100 μM in the first column. Twoplates were prepared using 20 μl volumes of a double dilution series ofmixtures consisting of:

-   -   1. 10 μl of 10×LSE mixed with 10 μl of 100 μM carboplatin.    -   2. 10 μl of 10×LSE mixed with 10 μl of 100 μM irinotecan.

All plates were incubated at 37° C. in free-CO₂ humidified atmospherefor 5 days. The antiproliferative or the cytotoxic effect of leechsaliva extract was performed using a CELLTITER-GLO luminescent cellviability assay based on measuring the luminescence signal from thereaction between the ULTRA-GLO recombinant luciferase and the ATPmolecules produced by the metabolically viable cells in the presence ofMg⁺² and molecular oxygen (from Promega, 2009). A CELLTITER-GLO assaywas performed according to standard protocols:

-   -   1. CELLTITER-GLO reagent was prepared by mixing CELLTITER-GLO        buffer and the substrate which were previously equilibrated to        room temperature.    -   2. The 5-day incubated 96-well plates containing the        experimental cells were allowed to be equilibrated to room        temperature prior the assay. The medium was aspirated from all        wells and replaced by 100 μl of new CGM.    -   3. An equal volume of the prepared CELLTITER-GLO reagent (100        μl) was pipetted into the well, and then mixed for 2 min using        the orbital plate shaker and let to stand at room temperature        for 10 min to stabilize the luminescent signal.    -   4. The reaction medium (CGM+CELLTITER-GLO reagent) was        transferred into new white 96-well plate suitable for the        luminometer used.    -   5. Luminescence was recorded by the luminometer.    -   6. Cell inhibition was calculated from the equation (Xu, Guo,        Li, Wei, & Zhao, 2008):

${\%\mspace{14mu}{inhibition}} = {\frac{{{Control}\mspace{14mu}{signal}} - {{Sample}\mspace{14mu}{signal}}}{{Control}\mspace{14mu}{signal}} \times 100}$

-   -   7. The concentration of the test sample (LSE or the negative        control) which inhibits 50% of cell growth (IC₅₀) was averaged        from three replicates and estimated from plotting the percentage        of cell growth inhibition against test sample concentration.        (Hsu et al., 2011). Plots were carried out using a Four        Parametric Logistic Equation using Sigma Plot 11.0 software.

After an incubation period of 5 days at 37° C. in CO₂-free humidifiedenvironment, the cells reached almost 90% confluence. The cells wereharvested by detaching them from the cell culture flask walls withACCUTASE and centrifuged at 4° C. and 125×g for 10 minutes. Cellcounting with a trypan blue method revealed that one flask containsapproximately 5.550-5.740×10⁶ viable cells at near 90% confluence.

FIG. 20 shows that the LSE showed remarkable anti-proliferation activityagainst human small cell lung cancer (SW1271 cell line), according tosome embodiments. The concentration of total LSE protein that inhibitsgrowth of 50% of the cells after 5 days incubation (IC₅₀) was 119.844μg/ml, estimated by plotting percent inhibition against total proteinconcentration. The phagostimulatory solution alone had no effect on cellproliferation.

FIGS. 21 and 22 show the cytotoxic effect of mixtures of LSE withirinotecan or carboplatin, according to some embodiments. The IC₅₀ ofirinotecan and carboplatin were 5.813 μg/ml and 18.754 μg/ml,respectively. All measurements were repeated in triplicate, and themean±the standard error of the mean SEM (n=3) were considered. Plotswere generated using Four Parametric Logistic Equation with Sigma Plot11.0 software.

A combination of LSE and irinotecan showed an IC_(50comb) of 51.463μg/ml which is about 57.1% less than the IC₅₀ of LSE used alone. Acombination of LSE and carboplatin show an IC_(50comb) of 114.261 μg/ml,a 4.6% reduction in IC₅₀. Carboplatin showed a dramatic decline in IC₅₀value by 65%, such that IC_(50comb) of carboplatin and LSE was 6.449μg/ml.

The results suggest that LSE, alone or in combination with other agentssuch as irinotecan or carboplatin could be useful in treating otherforms of cancer, such as prostate, breast, and liquid cancers such asleukemias and lymphomas. Acute myeloid leukemia is of particularinterest.

Example 5 A Method of Treating a Diabetes

This example shows the effectiveness of LSE in treating diabetes. TheLSE isolation and total protein measurement was done according to themethods taught herein.

Materials and Methods:

Sodium chloride, arginine hydrochloride, absolute ethanol andformaldehyde 37% (from Merck); Bradford reagent kit (Amresco Inc.);Parafilm membrane (from American Can Company); anhydrous glucose (fromFisher Scientific); alloxan monohydrate used to induce diabetes (fromSigma Aldrich); bovine serum albumin (from Sigma Aldrich); insulin frombovine pancreas (≧27 units/mg)(from Sigma Aldrich); the method ofpreparing Alloxan solution in ice-cold normal saline immediately priorto injection (from Lenzen, 2008).

Centrifugation was done using Universal 32R centrifuge (from HettichZenTrifugen, Germany); microplate reader model INFINITE M200, NANOQUANTTECAN (from TECAN USA); lyophilization performed using a CHRISTfreeze-drier model Alpha 1-4LD (Germany); a ONE TOUCH ULTRA glucometerand test strips used for the determination of blood glucoseconcentration (from LifeScan Inc., USA); and, a NIKON ECLIPSE 80imicroscope.

Male rats of Sprague Dawley strain (SD) (from Mikro Makmur Enterprise,Kuantan, Pahang Darul Makmur, Malaysia) were grouped randomly and keptat an animal post-graduate laboratory in Kulliyyah of Pharmacy,International Islamic University Malaysia (IIUM), maintained with an airconditioning system and exhaust fans. The rats were under a 12 h/12 hdark and light cycle at room temperature (25° C.). They wereacclimatized to these conditions for one week prior to the experimentand housed in polypropylene cages lined with pine wood husk changedevery two days. The rats were given free access to tap water and acommercial dry pellet diet, Gold Coin. The experimental procedures wereconducted according to Principles and Guide to Ethical Use of LaboratoryAnimals approved by Ministry of Health Malaysia (Sinniah & Hussein,2000). The experimental protocols were approved by Ethics CommitteeMeeting (No. 1/2011 on 22 Apr. 2011) of Kulliyyah of Medicine, IIUM(Ref. No. IIUM/305/20/4/10).

The rats were fasted overnight and a type-1-like diabetes was induced bya single-dose intraperitoneal (i.p) administration of freshly preparedalloxan solution 160 mg/kg body weight (b.w.) (Rajakopal & Sasikala,2008). In order to prevent fatal alloxan-induced hypoglycemia, the ratswere administered a 20% glucose solution intraperitoneally followed by a5% glucose solution orally for the next 12 hours (Lenzen, 2008). Therats were then fed a commercial pellet diet ad libitium and given freeaccess to tap water. All experimental animals were injected three timeswith alloxan at 24 hours intervals.

After 24 hours of alloxanisation, the fasting blood glucoseconcentration (FBG) was measured every morning to check the diabeticstate of the injected rats. All FBG values were taken from freshcapillary blood from a tail vein puncture, and measurements were takenusing a ONE TOUCH ULTRA glucometer. After three days of alloxaninjection, the rats showed FBG levels of more than 11.1 mmol/L, a levelconsidered diabetic for the study (Abeeleh et al., 2009).

Forty male rats were divided randomly into eight groups, each comprisingfive rats as detailed below:

-   -   Group I: normal control rats, neither alloxan nor LSE was        injected into this group.    -   Group II: induced-diabetic control rats injected only with        alloxan solution i.p    -   Group III: induced-diabetic rats injected subcutaneously (s.c)        with LSE 500 μg/kg b.w which corresponds to the protein        amount/dose given by one leech.    -   Group IV: induced-diabetic rats injected s.c with LSE 1000 μg/kg        b.w which corresponds to the protein amount/dose given by two        leeches.    -   Group V: induced-diabetic rats injected s.c with the        phagostimulatory solution PhS1 (0.001M arginine in normal        saline) in a dose of 20 ml/kg b.w which contains the amount of        the arginine and sodium chloride that is supposed to accompany        the higher dose of LSE injected to the group IV.    -   Group VI: induced-diabetic rats injected s.c with 20 units/kg        b.w bovine pancreas insulin suspension in distilled water (Booth        & Brookover, 1968).    -   Group VII: induced-diabetic rats injected s.c with 10 units/kg        b.w bovine pancreas insulin suspension in distilled water (Booth        & Brookover, 1968).    -   Group VIII: induced-diabetic rats injected s.c with 250 μg/kg        b.w LSE+10 units/kg b.w bovine pancreas insulin.

The antihyperglycemic activity of LSE was assessed by the fall in FBGvalues within eight hours. Fasting blood glucose (FBG) at two-hourintervals was recorded during the experiment period for all theexperimental animals. The percentage decrease in fasting blood glucoseconcentration was calculated from the following equation (Madubunyi,Onoja, & Asuzu, 2010):

${{Percentage}\mspace{14mu}{decrease}\mspace{14mu}{in}\mspace{14mu}{FBG}} = {\quad\left\lbrack {\frac{{{FBG}\mspace{14mu}{before}\mspace{14mu}{treatment}\mspace{14mu}\left( {0\mspace{14mu}{hr}} \right)} - {{FBG}\mspace{14mu}{after}\mspace{14mu}{treatment}\mspace{14mu}\left( {x\mspace{14mu}{hr}} \right)}}{{FBG}\mspace{14mu}{before}\mspace{14mu}{treatment}\mspace{14mu}\left( {0\mspace{14mu}{hr}} \right)} \times 100} \right\rbrack}$

Thirty male rats were divided randomly into six groups, each comprisingfive rats as detailed below:

-   -   Group A-I: rats injected i.p with a single dose of alloxan (160        mg/kg b.w).    -   Group A-II: rats injected i.p with two doses of alloxan (160        mg/kg b.w) at 24-hour interval.    -   Group A-III: rats injected i.p with three doses of alloxan (160        mg/kg b.w) at 24-hour interval.    -   Group A-IV: rats injected s.c with a single dose of LSE (250        μg/kg b.w) followed after one hour by a single dose i.p of        alloxan (160 mg/kg b.w).    -   Group A-V: rats injected s.c with two doses of LSE (250 μg/kg        b.w) followed after one hour by two i.p doses of alloxan (160        mg/kg b.w) at 24-hour interval.    -   Group A-VI: rats injected s.c with three doses of LSE (250 μg/kg        b.w) followed after one hour by three i.p doses of alloxan (160        mg/kg b.w) at 24-hour interval.

The prophylactic activity of LSE was assessed by measuring FBG after 24hours of each injection. Rats that exhibited FBG values between 8.3 and13.9 mmol/L were considered as mild diabetic and those with FBG of morethan 13.9 mmol/L were considered as severe diabetic (Gupta et al.,2009).

FIGS. 23 and 24 show the effect of different doses of LSE and insulin onfasting blood glucose (mmol/L) in normal and diabetic rats at varioustime intervals (h), according to some embodiments. After three days ofintraperitoneal alloxan injection (160 mg/kg b.w), the FBG increasedsignificantly (p<0.001) in the FBG in the diabetic control rats whencompared with the normal control ones. The FBG levels were significantlyreduced after injecting rats with LSE subcutaneously at both doses of1000 and 500 μg/kg b.w. The LSE at a dose of 1000 μg/kg b.w resulted ina higher significant decline (p<0.001) in FBG than that of the dose 500μg/kg (p<0.05). In addition, a significant reduction in FBG (p<0.05)occurred after two hours of injection with LSE, and a higher significantdecline was noticed after four, six and eight hours (p<0.001).

All insulin-injected rats experienced a sharp significant decrease(p<0.001) in FBG after two hours of injection. Rats injected with thelower dose of insulin (10 units/kg b.w) returned to the diabetic statewith rapid increasing FBG values after 8 hours of treatment. Rats thatreceived insulin (10 units/kg) and LSE (250 μg/kg) exhibited asignificant drop in FBG during the whole 8-hour study period. Diabeticrats injected with the phagostimulatory solution showed no significantreduction in FBG compared with the diabetic control group. Nosignificant difference was seen between the normal rats and the diabeticrats treated with LSE or insulin at either dose.

Neither mortality nor a behavioural change was observed amongst the allsaliva-injected animals until the end of the study. All these animalsexhibited typical locomotion and physical activity, such as no signs ofweakness or aggressiveness. No toxicity reaction were noticed forexample no anorexia, ataxia, piloerection, loss of weight, diarrhoea,urination, breathing difficulty and noisy breathing. Simultaneousadministration of LSE (250 μg/ml b.w) and insulin (10 units/kg b.w)induced hypoglycemia with no mortality. No signs of acute toxicity wereobserved in rats injected subcutaneously with LSE at both doses of 1000and 500 μg/kg b.w, On the other hand, injection insulin at a dose of 20units/kg b.w resulted in a hypoglycemic condition in all rats leading tothe death of one rat. The other rats which stayed alive showed lessphysical activity especially during the first two hours of injection.

FIG. 25 shows that the LSE has a prophylactic effect on the onset ofdiabetes, according to some embodiments. For example, without use of LSEas a prophylactic, one dose of alloxan (160 mg/kg b.w) was not enough toinduce diabetes in the rats, two doses made all rats mildly diabetic(10.1±2.0 mmol/L), and three doses induced severe diabetes (27.3±2.1mmol/L). None of the rats injected with one dose of LSE (250 μg/kg b.w)before alloxanisation got diabetic at all. Two doses of LSE were able toprevent diabetic induction in all rats when injected one hour before thealloxan injection. Only rats which received three doses of alloxan afterthree doses of LSE became mild diabetic (11.5±0.6 mmol/L). The resultsare the mean of triplicates±the standard error of the mean SEM (n=5),analyzed using one-way ANOVA and Tukey's HSD post hoc test; p<0.05 wasconsidered statistically significant. P<0.05 when compared with ratsinjected with two doses of alloxan and LSE. p<0.001 when compared withrats injected with three doses of alloxan and LSE.

Example 6 A Method of Treating a Viral Disease

This example will be used to show the effectiveness of LSE at treating aviral disease. The LSE isolation and total protein measurement will bedone according to the methods taught herein.

A tissue culture generally a chicken embryo 3-6 days old will beinfected in side the embryo membrane by a dose of live viruses(hepatitis C, HIV, Dengue, West Nile, and Influenza H1N1, H5N1) ifnecessary a multiple infections will be used until the infection takesplace.

After a period of incubation, embryo growth and changes in their tissueswill be monitored according to established procedures. This infectedtissue culture will serve as a control. Three other cultured tissueswill be prepared by the same method. In the first one, four doses of LSE(100/250/500/1000 ug/b.w) will be injected before the viral infection.In the second tissue culture, four doses of LSE (100/250500/1000 ug/b.w)will be injected at 1, 2, 3, 4 hours after the viral infection. In thirda tissue, four doses of inactivated LSE (100/250/500/1000 ug/b.w), willbe injected after and before infection. The aim in the third experimentis to analyze any change in signal pathways and comparison with theactive LSE will be made in order to have an indication of the mechanismof action. The three cultured tissues: treated after and before and withinactivated LSE will be monitored and compared to the control tissue.

The effect LSE on viral infection will be then deduced according toestablished procedures and will be compared to the conventional drugslike interferon.

Example 7 A Method of Treating a Parasitic Disease

This example will be used to show the effectiveness of LSE at treating aparasitic disease. The LSE isolation and total protein measurement willbe done according to the methods taught herein.

In the case of malaria, an animal model, generally a mouse model will beinjected by a dose of a malaria parasite (the dose will be determinedaccording to the virulence of the parasite Spp) until the animalsdevelop the desired symptoms of malaria. The sick animals will beinjected (SC or IV mode) by two doses (500/1000 ug/K b.w LSE). Aftertreatment, blood drops will be taken from the mice tail on glass slidesfor examination under the microscope, or for ELIZA testing.

We will use eight groups of ten mice each as follows:

-   -   1. Group A—control infective positive    -   2. Group B—Infected & treated with LSE (500 ug/K bw) after 1, 2,        3, 4. hours    -   3. Group C—Infected & treated with LSE (1000 ug/K bw) after 1,        2, 3, 4. hours    -   4. Group D—½ hour before the infection with the parasite we        inject it with LSE (500 ug/K bw) as prophylactic measures.    -   5. Group E—½ hour before the infection with the parasite we        inject it with LSE (1000 ug/K bw) as prophylactic measures.    -   6. Group F & G—Infected mice injected with 500 & 1000 ug/K bw of        phagostimulatory solution.

Blood smears will be taken from the tail of the mice daily and examinedunder a light microscope for seven days to monitor the activity of theLSE. The last (H) infected group will be treated with a standardantimalaria drug to monitor the relative activity and potency of LSE onthe parasite.

Example 8 A Method of Administering an Antioxidant Therapy

This example shows the antioxidant activity of LSE. The LSE isolationand total protein measurement was done according to the methods taughtherein.

Materials and Methods

Methanol (MeOH) (from Fischer Scientific); L-Ascorbic acid (fromCalbiochem); arginine hydrochloride, bovine serum albumin (BSA), andDPPH (2,2-Diphenyl-1-picrylhydrazyl) (from Sigma Aldrich); sodiumchloride (NaCl) (from Merck).

An Infinite M200, NanoQuant TECAN multi detection microplate reader(from TECAN (USA)); a Hettich ZenTrifugen Universal 32R centrifuge(Germany); a CHRIST freeze-drier model Alpha 1-4LD (Germany).

Antioxidant activity was determined using known methods (Blois, 1958) ofmeasuring radical scavenging ability using 2,2-diphenyl-1-picrylhydrazyl(DPPH) (Althunibat et al., 2009; Blois, 1958; Sanja, Sheth, Patel,Patel, & Patel, 2009):

-   -   1. Preparation of DPPH solution: a DPPH solution (0.002M) was        prepared by dissolving 4.3 mg DPPH in 3.3 ml MeOH. The resultant        solution was protected from light by covering the container with        an aluminum foil.    -   2. Preparation of test sample: The lyophilized LSE was dissolved        in the minimum amount of distilled water (2 ml). The volume was        brought to a final volume of 6.6 ml by MeOH yielding a 3-time        concentrated LSE, and the resultant methanol solution was termed        as 3×mLSE. Volumes of 100 μl of serial double-fold dilutions of        the 3×mLSE were pipetted into a 96-well plate. All volumes were        brought to a final volume of 300 μl by MeOH.    -   3. Preparation of standard solution: A stock solution of        ascorbic acid (50 μg/ml) was prepared by dissolving 500 μg of        ascorbic acid in 10 ml MeOH with vigorous shaking. Serial        step-wise dilutions were prepared in the 96-well plate by taking        different volumes of the stock solution and diluting them with        MeOH up to 300 μl, corresponding to final concentrations of 50,        40, 30, 20, 10, 5 and 2.5 μg/ml.    -   4. Experimental protocols: A volume of 15 μl of DPPH solution        (0.002M) was added to 300 μl of MeOH. Immediately, the        absorbance at 516 nm (A₅₁₆) was measured as a control reading.        15 μl of DPPH solution was added to the test samples and        standard solutions. The A₅₁₆ of the test samples were taken        after 15 min, and the same procedures were used with PhS1 as a        negative control.    -   5. Calculations: The free radical scavenging activity (%        antiradical activity) was estimated from the equation:

${\%\mspace{14mu}{antiradical}\mspace{14mu}{activity}} = {\frac{{{Control}\mspace{14mu}{absorbance}} - {{Sample}\mspace{14mu}{absorbance}}}{{Control}\mspace{14mu}{absorbance}} \times 100}$

FIGS. 26 and 27 compares the free radical scavenging activity of LSE toL-ascorbic acid (vitamin C), according to some embodiments. Allmeasurements were repeated in triplicate, and the mean±SEM wasconsidered. The concentration of ascorbic acid and the LSE proteins(μg/ml) required for scavenging 50% of DPPH (IC₅₀) was estimated fromthe curve resulted from plotting % antiradical activity againstconcentrations (μg/ml). Plots were carried out using Four ParametricLogistic Equation using Sigma Plot 11.0 software.

A dose-dependent free radical scavenging activity was shown by the LSEhaving an IC₅₀ of 7.282 μg/ml. Similarly, L-ascorbic acid was found tobe a free radical scavenger with IC₅₀ of 5.803 μg/ml.

Example 10 A Method of Administering an Antibacterial Therapy

This example shows the effectiveness of LSE as an antibacterial.

Methods and Materials

Mueller-Hinton agar (MHA) and Mueller-Hinton broth (MHB) (from OxoidLtd.); potato dextrose agar (PDA) and potato dextrose broth (PDB) (fromLiofilchem); antimicrobial susceptibility test discs containing 5μg/disc ciprofloxacin and 100 units/disc nystatin (from Oxoid Ltd);bovine serum albumin and arginine hydrochloride (from Sigma-Aldrich);sodium chloride (NaCl) (from Merck); Bradford reagent kit (fromAmresco).

a Laminar Flow Hood Jouan (Jouan SA, France); an incubator Memmert/INB400 and water bath Memmert/WNB 22 (from Memmert GmbH, Germany); aHIRAYAMA/HV-85 Autoclave (HIRAYAMA Corporation, Japan); a HITACHI U-1900Spectrophotometer (from HITACHI High-Tech (Japan)); sterile 96-wellplates (from Greiner Bio-One Corporation): a centrifuge HettichZenTrifugen Universal 32R (Germany).

Reference strains of human pathogens were used including Gram-positivebacterial spp. (Bacillus cereus ATCC25923 and Staphylococcus aureusATCC25923), Gram-negative bacterial strains (Pseudomonas aeruginosaATCC27853, Escherichia coli ATCC35218 and Salmonella typhi fromInstitute of Medical Research Health Ministry IMR) and two fungalstrains (Candida albicans ATCC10231 and Cryptococcus neoformansATCC90112).

All media used during the experimental procedures were preparedaccording to the manufacturer instructions, as the following:

-   -   1. Muller-Hinton agar (MHA): was prepared by suspending 38 g of        MHA in 1 L distilled water with boiling and frequent vigorous        agitation until completely dissolved. Then, it was sterilized by        autoclaving at 121° C. for 15 minutes.    -   2. Muller-Hinton broth (MHB): was prepared by suspending 21 g of        MHB in 1 L distilled water with boiling and frequent vigorous        agitation until completely dissolved. Then, it was sterilized by        autoclaving at 121° C. for 15 minutes. The resultant stock        sterile broth was kept in a well closed 1000-ml screw-cap        bottle, wrapped with parafilm membrane at the cap and stored at        +4° C. for further usage. Before usage, MHB was warmed (37° C.)        in the incubator for 15 minutes.    -   3. Potato dextrose agar (PDA): was prepared by suspending 39 g        of PDA in 1 L distilled water with boiling and frequent vigorous        agitation until completely dissolved. Then, it was sterilized by        autoclaving at 121° C. for 15 minutes.    -   4. Potato dextrose broth (PDB): was prepared by suspending 26.5        g in PDB 1 L distilled water with boiling and frequent vigorous        agitation until completely dissolved. Then, it was sterilized by        autoclaving at 121° C. for 15 min. The resultant stock sterile        broth was kept in a well closed 1000-ml screw-cap bottle,        wrapped with parafilm membrane at the cap and stored at +4° C.        for further usage. Before usage, PDB was warmed (37° C.) in the        incubator for 15 minutes.

Before pouring of agar, the freshly prepared sterilized agar mediumMHA/PDA for bacterial/fungal strains, was allowed to cool in water bathadjusted at 50° C. for 15-30 minutes in order to prevent the formationof moisture droplets by condensation phenomenon. Thereafter, a volume ofabout 20-25 ml was poured into disposable flat-bottom sterile(gamma-irradiated) Petri dishes to a height of 4 mm avoiding trappingany air bubbles. The plates with lids ajar were left to equilibrate atroom temperature for about 15 minutes under the laminar flow to get ridof excess surface moisture and temperature. Finally, the plates werecovered, inverted downside upwards and stored in the refrigerator (+4°C.) to be used within a maximum period of one week. Before inoculationof the agar-containing plates, they were equilibrated to roomtemperature for about one hour in order to minimize condensation (Coyle,2005; Goldman & Green, 2009; Lalitha, 2004).

A turbid-metric assay was carried out to standardize the microorganismnumber used for inoculation. The Direct Colony Suspension method wasused to prepare inoculation suspension (Coyle, 2005; Goldman & Green,2009; Lalitha, 2004; Rex, Pfaller, Rinaldi, Polak, & Galgiani, 1993).Experimental procedures include the following steps:

-   1. Preparation of Barium Sulfate (0.5 McFarland) standard    suspension: It was prepared by adding 0.5 part of 0.048M BaCl₂ to    99.5 parts of 0.18M H₂SO₄ and agitated vigorously until a homogenous    suspension was obtained. The turbidity of the suspension was    verified by measuring the optical density at 625 nm (OD₆₂₅) by the    spectrophotometer. Proper dilutions were done to get an absorbance    value of 0.008-0.10 which corresponds to 0.5 McFarland standards.    The prepared standard suspension was aliquoted into small screw-caps    glass bottles, stored at room temperature and protected from light.    Before utilization, the bottles were stirred well by vortex to    maintain a uniform suspension.-   2. Direct Colony Suspension method:    -   a) Under aseptic condition, five colonies isolated by        ignition-sterilized inoculation loop from 18-24-hour cultivated        agar plates of each microbe were suspended separately in 20 mL        pre-warmed (37° C.) broth medium (MHB for bacterial strains or        PDB for fungi stains) kept in screw-cap bottles and incubated at        37° C.    -   b) During the incubation period, aliquots of 1 mL were taken        from the culture at hourly intervals and the optical density        (OD₆₂₅ for bacterial suspension and OD₅₃₀ for fugal suspension)        were measured using the spectrophotometer.    -   c) Proper dilutions by MHB/PDB were done in order to adjust the        microorganism suspension to match the 0.5 McFarland turbidity        standards. Broth suspension and the Barium Sulfate 0.5 McFarland        standard were compared by the naked eye against a card with a        white background and black lines. Finally, the resultant broth        suspensions contained 10⁷ CFU/ml for bacterial spp. and 10⁴        CFU/ml for fungal spp. which were used for all experiments        performed.    -   d) Suspensions were always agitated thoroughly before OD        measurement and inoculation.

The Kirby-Bauer Disc Diffusion method was used for determiningantimicrobial activity of LSE:

-   1. Inoculation of test plates: a sterile cotton swab was immersed    and rotated many times in the adjusted microorganism suspension. The    immersed swab was pressed strongly till all excess fluids were    removed. The swab was passed over the dried sterile agar surface of    MHA-containing plates for bacterial strains or PDA-containing plates    for fungal strains. These steps were repeated three times by    spreading the broth suspension using glass rod in order to get    evenly inoculated plates. The plates were left open under the    laminar flow hood for 5 minutes to allow the surface to absorb the    extra moisture.-   2. Preparation of dried filter paper discs: WHATMAN filter paper No.    1 was used to prepare discs approximately 6 mm in diameter. These    discs were dipped in test solution (LSE or PhS1, steriled by    filtration through 0.2 μm sterile SARTORIUS filter paper).    Thereafter, they were allowed to dry for 5 minutes in the incubator    (37° C.) before application onto the Petri dishes. The discs were    left at room temperature for about 15 minutes before placement to    the inoculated agar Petri dishes.-   3. Discs placement to the inoculated agar Petri dishes: The filter    discs loaded with test solution (LSE/PhS1) and reference    antibiotic-containing discs (5 μg/disc ciprofloxacin or 100    units/disc nystatin) were laid down on the inoculated agar plates    using sterile forceps with gentle pressing to ensure a good    adherence to the agar surface. The discs were distributed to be at    least 15 mm from the edge of the plate and no closer than 24 mm from    center to center. Lastly, the plates were inverted upside downward    and incubated at 37° C. for 24 h and 48 h for bacterial and fungal    spp., respectively.

After the incubation period, the zone of inhibition (mm) around eachdisc was measured using a ruler and compared with the referenceantibiotics used.

Microdilution was used to determine the minimal inhibitory concentration(MIC), which is the minimal concentration of LSE protein content thatcan inhibit the growth of the test organism. Serial double-folddilutions of LSE were carried out in a sterile 96-well plate. 100 μl ofsterile Mueller-Hinton broth was pipetted into the first five columnswells of the plate. 100 μl of LSE was mixed with the broth in the firstthree wells of the first row of the plate. Dilutions were made bytransferring 100-μl aliquots of the mixture from the first three wellsinto the next ones vertically, and so forth. 10 μl of the test organismsuspension broth containing (10⁷ CFU/ml) was pipetted into each well ofthe first four columns, but no inoculum was added to the fifth column.The fourth and the fifth columns were considered as positive (broth withinoculum) and negative control (broth only), respectively. The plate wascovered, wrapped with parafilm sheets around the edges to avoiddehydrating, and incubated for 24 hours at 37° C. After the incubationperiod, the MIC endpoint was determined by a lack of turbidity in thewell.

The antibacterial and antifungal activities of the fresh LSE and thelyophilized samples are shown in Table 3 with ciproflaxin and nystatin,where the LSE is shown to have desired activity.

TABLE 3 Bacterial spp. Fungal spp. Starvation S. P. Sal. E. B. C. C.period aureus auroginosa typhi coli cereus albicans neoformance Sample(weeks) Zone of inhibition (mm) Fresh 16 — 0 22 25 0 — — LSE 22 0 0 0 00 — — 26 0 0 0 0 0 — — Lyoph.  22^(a) 11 0 10 0 0 0 0 LSE  26^(b) 0 0 00 0 0 0 Arginine + — — 0 0 0 0 0 0 NaCl^(c) Cipro — 24 24 35 26 28 — —(5 μg)^(d) Nystatin — — — — — — 10 9 (100 unit)^(e) ^(a)five foldsconcentrated, ^(b)ten folds concentrated, ^(c)the phagostimulatorysolution as negative control, ^(d, e)reference antibiotics, 0: noinhibition, —: not determined

Example 11 A Method to Test, Solid and Liquid Tumor Cell Types

This example discusses an in vitro assessment of the activity of LSE asapplied to cancer and non-cancer cell lines.

In order to test the LSE for it's anticancer efficacy, it can be appliedto additional cell lines that include, for example, MCF-7 (breast), PC-3(prostate), K562 (leukemia), MeWo (skin melanoma), Mia PaCa-2(pancreatic carcinoma), A549 (lung cancer), U87MG (brain tumor,glioblastoma), MCF10A (normal epithelial cells), HT-29 (coloncarcinoma), CaCo-2 (normal intestinal epithelial cells), HEP 3B (humanhepatoma liver cancer), ES-2 (ovarian carcinoma), HBEpC (normal humanepithelial cells), CCRF-CEM (leukemia), HL-60(TB) (leukemia), MOLT-4(leukemia), RPMI-8226 (leukemia), SR (leukemia), EKVX (non-small celllung), HOP-62 (non-small cell lung), HOP-92 (non-small cell lung).NCI-H226 (non-small cell lung), NCI-H23 (non-small cell lung), NCI-H322M(non-small cell lung), NCI-H460 (non-small cell lung), NCI-H522(non-small cell lung), COLO 205 (colon), HCC-2998 (colon), HCT-116(colon), HCT-15 (colon), KM12 (colon), SW-620 (colon), SF-268 (CNS),SF-295 (CNS), SF-539 (CNS), SNB-19 (CNS), SNB-75 (CNS), U251 (CNS), LOXIMVI (melanoma), MALME-3M (melanoma), M14 (melanoma), SK-MEL-2(melanoma), SK-MEL-28 (melanoma), SK-MEL-5 (melanoma), UACC-257(melanoma), UACC-62 (melanoma), IGR-OVI (ovarian), OVCAR-3 (ovarian),OVCAR-4 (ovarian), OVCAR-5 (ovarian), OVCAR-8 (ovarian), SK-OV-3(ovarian), 786-0 (renal), A498 (renal), ACHN (renal), CAKI-1 (renal),RXF-393 (renal), SN12C (renal), TK-10 (renal), UO-31 (renal), DU-145(prostate), NCl/ADR-RES (breast), MDA-MB-231/ATCC (breast), HS 578T(breast), MDA-MB-435 (breast), MDA-MB-468 (breast), BT-549 (breast),T-47D (breast), Saos-2 (bone cancer).

Materials and Methods

a 96 well flat bottom plate; MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigmacatalogue #M2128); reagent reservoirs; a multi-channel pipette; amultichannel repeater pipette; a set of single pipettors, 10 μL, 200 μL,1000 μL; and various pipette tips.

Growth media, reagents and serum including Dulbecco's Modified EagleMedia (DMEM), high glucose; RPMI-1640 media; Iscove's; Hank's BufferedSalt Solution; L-glutamine; Fetal Bovine Serum; and Trypsin/EDTA.

To perform the MTT cytotoxicity assay, produce a stock solution of 5 mgMTT/ml PBS (phosphate buffered saline); use a sterile filter with 0.22μM syringe filter; and store at 4° C. in the dark. Produce a workingsolution of 1 mg/ml dilute MTT stock solution with 1:4 (v/v) inpre-warmed culture medium.

Day 1, plate cells; Day 2, add drugs; Days 3-5, Read plates.

An example of a plate array design is as follows:

1 2 3 4 5 6 7 8 9 10 11 12 A X X X X X X X X X X X X B X media control1433 717 358 179 90 45 22 11 X C X media control 1433 717 358 179 90 4522 11 X D X media control 1433 717 358 179 90 45 22 11 X E X mediacontrol 1433 717 358 179 90 45 22 11 X F X media control 1433 717 358179 90 45 22 11 X G X media control 1433 717 358 179 90 45 22 11 X H X XX X X X X X X X X X X: H₂O; final volume all wells: 200 μL

Day 1: Plating Cells

Two cell lines can be plated on one 96-well plate such that tests arecompleted in triplicate for each (K562 is non-adherent, for example, andso should be on separate plate). Adherent cell lines must be plated oneday prior to adding drugs to allow the cells to adhere to the plate,whereas non-adherent cells (suspension cells) can be plated on the sameday (Day 2) prior to adding drugs.

Cells are first counted on the hemocytometer to observe the viability ofthe cells which should be greater than or equal to 90%. If this is notthe case, the live cells should be separated from the dead cells usingFicoll-Paque (3 mL per 4 mL of cells, centrifuge for 25 min at 1500 rpm,wash cells once with culture medium 5 min at 1500 rpm). The number ofcells to be plated per well varies with the growth rate of the cellline. The ideal optical density (O.D.) of the control cells should bebetween 1.00 and 2.00 by the end of the incubation time. Due toincreased rate of evaporation along the border of the wells of theplate, it is not used for the assay, but is filled with 200 μl ofsterile water.

Column B2-G2 is used as the blank and is filled with 200 μl of medium.Column B3-G3 is the control column with 100 μl of cells only. Drug isadded to the cells in triplicate, therefore columns B4-D4, B5-D5, B6-D6etc. to B11-D11 allows for 8 different concentrations of drug to betested. This applied to the bottom half of the plate as well.

When adding cells to the plate to make sure that the cells are wellsuspended so that the same number of cells will be added to each well.Leave the plate in an incubator overnight for adherent cell lines tosettle on the plate.

Day 2: Adding Drugs

If there are adherent cells, aspirate the used medium and add 100 μl offresh medium. Add 100 μl of drug of desired concentration in each well.Since the total volume of the medium per well is 200 μl, dilute the drugaccordingly. If you want the final concentration of drug to be 10 μM inthe well, for example, you will have to make a 20 μM solution (100 μl ofthis plus 100 μl of your medium will give you a 10 μmol solution in atotal of 200 μl). Add 100 μl of medium to wells in the control column(B3-G3). Return plate to incubator 72 hours.

Reading the Plate

At the end of the incubation period, add 50 μl per well of MTT workingsolution to all of the wells. Return plate to incubator for 3-4 hoursand keep incubation times constant for repeat experiments. Set up yourassay template on the plate reader. After 3-4 hours, remove plates fromincubator. If the cells are non-adherent, they must be spun down for 10minutes at 1800 rpm. Tip the 96 well plate on a 45° angle towardsyourself. Using a 10-100 μl pipet tip attached to the vacuum, aspiratesupernatant from each well. Add 150 μl DMSO per well.

Resuspend cells by placing plate on the plate shaker (5-10 min should belong enough). If needed, resuspend tough cells by hand using themultichannel pipette, being careful not to create bubbles. The best wayto get rid of any bubbles in a 96 well plate is to direct a gentlestream of air onto the plate. Place plate into spectrophotometer andread at 570 nm.

Data Collection

The following records will be collected: growth cell optimization foreach cell line; cytotoxicity of LSE; and cell viability graphs.

We claim:
 1. A method of treating a bacterial skin infection of asubject in need thereof, comprising administering an effective amount ofa refined, whole leech saliva extract to the skin of the subject, theextract having a peptide molecular weight distribution with molecularweights of 3496 Daltons or greater; wherein the whole leech salivaextract functions as an agent in the treatment of the bacterial skininfection, and wherein the extract is produced from a processcomprising: feeding a phagostimulatory agent to a leech; inducing aregurgitation in the leech, the inducing including placing the leech inan environment having a temperature of less than 0° C. or about 0° C.;and, collecting an unrefined, whole saliva in the regurgitation of thecooled leech.
 2. The method of claim 1, further comprising revitalizingthe leech by warming the leech in a water bath having a temperatureranging from about 5° C. to about 40° C.
 3. The method of claim 1,wherein the method further comprises creating a refined, whole-salivaextract; the creating including removing solid components from theunrefined, whole saliva.
 4. The method of claim 1, wherein the methodfurther comprises lyophilizing separate volumes of the refined, wholesaliva extract, the volumes not exceeding about 2 ml each.
 5. The methodof claim 1, the leech being Hirudinaria manillensis.
 6. The method ofclaim 1, wherein the bacterial infection is selected from the groupconsisting of anaplasmosis, anthrax, botulism, brucellosis,campylobacteriosis, cellulitis, chancroid, chlamydia, chlamydiatrachomatis, cholera, diphtheria, donovanosis, ehrlichiosis, erysipelas,impetigo, shiga toxin- and verocytotoxin-producing Escherichia coli(STEC/VTEC) infection, Escherichia coli 0157:H7 infection, gonococcalinfection, gonorrhea, methicillin-resistant Staphylococcus aureusinfection (MRSA), Streptococcal infection, syphilis, tetanus, toxicshock syndrome, tularemia, vancomycin intermediate Staph aureus (VISA)infection, Staphylococcus pyogenes infections, vancomycin resistantStaph aureus (VRSA) infection, Shigella infection, and Salmonellainfection.
 7. The method of claim 1, wherein the bacterial infectionincludes a gram-positive bacteria.
 8. The method of claim 1, wherein thebacterial infection includes Bacillus cereus or Staphylococcus aureus.9. The method of claim 1, wherein the bacterial infection includes agram-negative bacteria.
 10. A method of treating a bacterial skininfection of a subject in need thereof, comprising administering aneffective amount of a refined, whole leech saliva extract to thesubject, the extract having a peptide molecular weight distribution withmolecular weights of 3496 Daltons or greater; wherein the whole leechsaliva extract functions as an agent in the treatment of the bacterialskin infection, wherein the extract is produced from a processcomprising: feeding a phagostimulatory agent to a leech; inducingregurgitation in the leech, the inducing including placing the leech inan environment having a temperature ranging from about −5° C. to about15° C.; collecting an unrefined, whole saliva in the regurgitation ofthe cooled leech; removing solid components from the unrefined, wholesaliva to create a refined, whole saliva; and, lyophilizing separatevolumes of the refined, whole saliva extract, the volumes not exceedingabout 2 ml each.
 11. The method of claim 10, wherein the bacterialinfection is selected from the group consisting of anaplasmosis,anthrax, botulism, brucellosis, campylobacteriosis, cellulitis,chancroid, chlamydia, chlamydia trachomatis, cholera, diphtheria,donovanosis, ehrlichiosis, erysipelas, impetigo, shiga toxin- andverocytotoxin-producing Escherichia coli (STEC/VTEC) infection,Escherichia coli 0157:H7 infection, gonococcal infection, gonorrhea,methicillin-resistant Staphylococcus aureus infection (MRSA),Streptococcal infection, syphilis, tetanus, toxic shock syndrome,tularemia, vancomycin intermediate Staph aureus (VISA) infection,Staphylococcus pyogenes infections, vancomycin resistant Staph aureus(VRSA) infection, Shigella infection, and Salmonella infection.
 12. Themethod of claim 10, wherein the bacterial infection includes agram-positive bacteria.
 13. The method of claim 10, wherein thebacterial infection includes Bacillus cereus or Staphylococcus aureus.14. The method of claim 1, wherein the bacterial infection includes agram-negative bacteria.
 15. The method of claim 1, wherein the molecularweight distribution of the whole saliva extract ranges from 4276 Daltonsto 44386 Daltons.
 16. The method of claim 1, wherein the molecularweight distribution of the whole saliva extract ranges from 6289 Daltonsto 14244 Daltons.
 17. The method of claim 1, wherein the molecularweight distribution of the whole saliva extract ranges from 10812Daltons to 88210 Daltons.
 18. The method of claim 1, wherein themolecular weight distribution of the whole saliva extract ranges from3496 Daltons to 88210 Daltons.